Nucleic acid products and methods of administration thereof

ABSTRACT

The present invention relates in part to nucleic acids, including nucleic acids encoding proteins, therapeutics and cosmetics comprising nucleic acids, methods for delivering nucleic acids to cells, tissues, organs, and patients, methods for inducing cells to express proteins using nucleic acids, methods, kits and devices for transfecting, gene editing, and reprogramming cells, and cells, organisms, therapeutics, and cosmetics produced using these methods, kits, and devices.

PRIORITY

This application is a continuation of U.S. patent application Ser. No. 15/748,132, filed Jan. 26, 2019, which is a U.S. National Stage (371) entry of PCT/US2017/047440, filed Aug. 17, 2017, which claims priority to U.S. Provisional Patent Application No. 62/376,209, filed Aug. 17, 2016 and to U.S. Provisional Patent Application No. 62/509,350, filed May 22, 2017. The entire contents of the aforementioned patent applications are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates, in part, to methods, compositions, and products for producing and delivering nucleic acids to cells, tissues, organs, and patients, methods for expressing proteins in cells, tissues, organs, and patients, and cells, therapeutics, and cosmetics produced using these methods, compositions, and products.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 11, 2019, is named FAB-010AC1_ST25.txt and is 2,439,673 bytes in size.

BACKGROUND

Synthetic RNA and Nucleic-Acid Therapeutics

Ribonucleic acid (RNA) is ubiquitous in both prokaryotic and eukaryotic cells, where it encodes genetic information in the form of messenger RNA, binds and transports amino acids in the form of transfer RNA, assembles amino acids into proteins in the form of ribosomal RNA, and performs numerous other functions including gene expression regulation in the forms of microRNA and long non-coding RNA. RNA can be produced synthetically by methods including direct chemical synthesis and in vitro transcription, and can be administered to patients for therapeutic use. However, previously described synthetic RNA molecules are unstable and trigger a potent innate-immune response in human cells. In addition, methods for efficient non-viral delivery of nucleic acids to patients, organs, tissues, and cells in vivo have not been previously described. The many drawbacks of existing synthetic RNA technologies and methods for delivery of nucleic acids make them undesirable for therapeutic or cosmetic use.

Cell Reprogramming and Cell-Based Therapies

Cells can be reprogrammed by exposing them to specific extracellular cues and/or by ectopic expression of specific proteins, microRNAs, etc. While several reprogramming methods have been previously described, most that rely on ectopic expression require the introduction of exogenous DNA, which can carry mutation risks. DNA-free reprogramming methods based on direct delivery of reprogramming proteins have been reported. However, these methods are too inefficient and unreliable for commercial use. In addition, RNA-based reprogramming methods have been described (see, e.g., Angel. MIT Thesis. 2008. 1-56; Angel et al. PLoS ONE. 2010. 5,107; Warren et al. Cell Stem Cell. 2010. 7,618-630; Angel. MIT Thesis. 2011. 1-89; and Lee et al. Cell. 2012. 151,547-558; the contents of all of which are hereby incorporated by reference). However, existing RNA-based reprogramming methods are slow, unreliable, and inefficient when performed on adult cells, require many transfections (resulting in significant expense and opportunity for error), can reprogram only a limited number of cell types, can reprogram cells to only a limited number of cell types, require the use of immunosuppressants, and require the use of multiple human-derived components, including blood-derived HSA and human fibroblast feeders. The many drawbacks of previously disclosed RNA-based reprogramming methods make them undesirable for research, therapeutic or cosmetic use.

Gene Editing

Several naturally occurring proteins contain DNA-binding domains that can recognize specific DNA sequences, for example, zinc fingers (ZFs) and transcription activator-like effectors (TALEs). Fusion proteins containing one or more of these DNA-binding domains and the cleavage domain of FokI endonuclease can be used to create a double-strand break in a desired region of DNA in a cell (see, e.g., US Patent Appl. Pub. No. US 2012/0064620, US Patent Appl. Pub. No. US 2011/0239315, U.S. Pat. No. 8,470,973, US Patent Appl. Pub. No. US 2013/0217119, U.S. Pat. No. 8,420,782, US Patent Appl. Pub. No. US 2011/0301073, US Patent Appl. Pub. No. US 2011/0145940, U.S. Pat. Nos. 8,450,471, 8,440,431, 8,440,432, and US Patent Appl. Pub. No. 2013/0122581, the contents of all of which are hereby incorporated by reference). Other gene-editing proteins include clustered regularly interspaced short palindromic repeat (CRISPR)-associated proteins. However, current methods for gene editing cells are inefficient and carry a risk of uncontrolled mutagenesis, making them undesirable for research, therapeutic or cosmetic use. Methods for DNA-free gene editing of somatic cells have not been previously explored, nor have methods for simultaneous or sequential gene editing and reprogramming of somatic cells. In addition, methods for directly gene editing cells in patients (i.e., in vivo) have not been previously explored, and the development of such methods has been limited by a lack of acceptable targets, inefficient delivery, inefficient expression of the gene-editing protein/proteins, inefficient gene editing by the expressed gene-editing protein/proteins, due in part to poor binding of DNA-binding domains, excessive off-target effects, due in part to non-directed dimerization of the FokI cleavage domain and poor specificity of DNA-binding domains, and other factors. Finally, the use of gene editing in anti-bacterial, anti-viral, and anti-cancer treatments has not been previously explored.

Accordingly, there remains a need for improved methods and compositions for the production and delivery of nucleic acids to cells, tissues, organs, and patients.

SUMMARY OF THE INVENTION

The present invention provides, in part, compositions, methods, articles, and devices for delivering nucleic acids to cells, tissues, organs, and patients, methods for inducing cells to express proteins, methods, articles, and devices for producing these compositions, methods, articles, and devices, and compositions and articles, including cells, organisms, cosmetics and therapeutics, produced using these compositions, methods, articles, and devices. Unlike previously reported methods, certain embodiments of the present invention provide small doses of nucleic acids to achieve significant and lasting protein expression in humans.

In some aspects, the present invention relates to a method for treating a metabolic disorder. The method comprises a step of administering an effective amount of a synthetic RNA encoding growth differentiation factor 15 (GDF15) to a subject in need thereof or endothelial cell specific molecule 1 (ESM1), wherein the synthetic RNA comprises one or more non-canonical nucleotides that avoid substantial cellular toxicity.

In various embodiments, the metabolic disorder is selected from Type I diabetes, Type II diabetes, insulin resistance, obesity, dyslipidemia, hypercholesterolemia, hyperglycemia, hyperinsulinemia, hypertension, hepatosteaotosis such as non-alcoholic steatohepatitis (NASH) and non-alcoholic fatty acid liver disease (NAFLD), cancer, a disease or disorder associated with impaired lipid metabolism, a disease or disorder associated with impaired renal function, a disease or disorder associated with impaired hepatic function, a disease or disorder associated with impaired lung function, a vascular or cardiovascular disease or disorder, muscle wasting, inflammation, and a respiratory disease.

In various embodiments, the disease or disorder associated with impaired renal function is selected from chronic kidney diseases, acute kidney injury, nephropathy, diabetic nephropathy, kidney failure, and kidney fibrosis.

In various embodiments, the vascular or cardiovascular disease or disorder is selected from coronary artery disease, cardiomyopathy, hypertension, atrial fibrillation, preeclampsia, peripheral artery disease, atherosclerosis, heart failure, acute myocardial infarction, acute coronary syndrome, muscle wasting, hypertensive ventricular hypertrophy, hypertensive cardiomyopathy, ischemic heart disease, myocardial infarction, abdominal aortic aneurysm, a blood clot, deep vein thrombosis, venous stasis disease, phlebitis, and varicose veins.

In various embodiments, the treatment results in reduction in one or more of aspartate transaminase (AST), alanine transaminase (ALT), or glucose levels in the subject, as compared to untreated subjects.

In various embodiments, the treatment results in increased lipid mobilization in the subject, as compared to untreated subjects.

In various embodiments, the administration is intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intracerebral, intravaginal, transdermal, intraportal, rectally, by inhalation, or topically.

In some aspects, the present invention relates to a method for modulating GDF15. The method comprises a step of administering an effective amount of a synthetic RNA encoding GDF15 to a subject, wherein the synthetic RNA comprises one or more non-canonical nucleotides that avoid substantial cellular toxicity.

In various embodiments, the modulating results in reduction in one or more of ALT, AST, or glucose levels in the subject, as compared to untreated subjects.

In various embodiments, the modulating results in increased lipid mobilization in the subject, as compared to untreated subjects.

In various embodiments, the administration is intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intracerebral, intravaginal, transdermal, intraportal, rectally, by inhalation, or topically.

In various embodiments, the modulating results in an increase in the quantity of GDF15 in the subject.

In various embodiments, the modulating results in a decrease in the quantity of GDF15 in the subject.

In some aspects, the present invention relates to a method for modulating ESM1. The method comprises a step of administering an effective amount of a synthetic RNA encoding ESM1 to a subject, wherein the synthetic RNA comprises one or more non-canonical nucleotides that avoid substantial cellular toxicity.

In various embodiments, the modulating results in one or more of reduced ALT, AST, or glucose levels in the subject, as compared to untreated subjects.

In various embodiments, the modulating results in increased lipid mobilization in the subject, as compared to untreated subjects.

In various embodiments, the administration is intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intracerebral, intravaginal, transdermal, intraportal, rectally, by inhalation, or topically.

In various embodiments, the modulating results in an increase in the quantity of ESM1 in the subject.

In various embodiments, the modulating results in a decrease in the quantity of ESM1 in the subject.

In some aspects, the present invention relates to a method for treating a liver disorder. The method comprises a step of administering an effective amount of a synthetic RNA encoding growth differentiation factor 15 (GDF15) or endothelial cell specific molecule 1 (ESM1) to a subject in need thereof, wherein the synthetic RNA comprises one or more non-canonical nucleotides that avoid substantial cellular toxicity; and the treatment reduces one or more of fatty liver, NAFLD, NASH, inflammation, hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma in the subject.

In various embodiments, the treatment results in reduction in one or more of AST, ALT, or glucose levels in the subject, as compared to untreated subjects.

In various embodiments, the treatment results in increased lipid mobilization in the subject, as compared to untreated subjects.

In various embodiments, the administration is directed to the liver.

In various embodiments, the administration is intraportal injection.

In some aspects, the present invention relates to a method for treating Friedrich's Ataxia. The method comprises a step of administering to a subject in need thereof an effective amount of (a) a synthetic RNA encoding a gene-editing protein capable of creating a double strand break in FXNA (SEQ ID NO: 582), and/or (b) a synthetic RNA encoding a gene-editing protein capable of creating a double strand break in FXNB (SEQ ID NO: 583), wherein the synthetic RNA comprises one or more non-canonical nucleotides that avoid substantial cellular toxicity. In various embodiments, the treatment comprises administering to a subject in need thereof an effective amount of (a) the synthetic RNA encoding the gene-editing protein capable of creating a double strand break in FXNA (SEQ ID NO: 582), and (b) the synthetic RNA encoding the gene-editing protein capable of creating a double strand break in FXNB (SEQ ID NO: 583).

In various embodiments, the administration is directed to the heart.

In various embodiments, the administration is via a catheter.

In various embodiments, the treatment ameliorates one or more of muscle weakness, loss of coordination, vision impairment, hearing impairment, slurred speech, scoliosis, pes cavus deformity of the foot, diabetes, and heart disorders, selected from one or more atrial fibrillation, tachycardia and hypertrophic cardiomyopathy.

In various embodiments, the gene-editing protein is selected from a CRISPR/Cas9, TALEN and a zinc finger nuclease.

In various embodiments, the gene-editing protein comprises: (a) a DNA-binding domain and (b) a nuclease domain. The DNA-binding domain comprises a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 629) and is between 36 and 39 amino acids long, wherein: “v” is Q, D or E; “w” is S or N; “x” is H, N, or I; “y” is D, A, I, N, G, H, K, S, or null; and “z” is GGKQALETVQRLLPVLCQD (SEQ ID NO: 630) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 631). The nuclease domain comprises a catalytic domain of a nuclease.

In various embodiments, the nuclease domain is capable of forming a dimer with another nuclease domain.

In various embodiments, the nuclease domain comprises the catalytic domain of a protein comprising the amino acid sequence of SEQ ID NO: 632.

In some aspects, the present invention relates to a method for reducing inflammation. The method comprises a step of administering an effective amount of a synthetic RNA encoding superoxide dismutase 3 (SOD3) or IFκB, wherein the synthetic RNA comprises one or more non-canonical nucleotides that avoid substantial cellular toxicity to a subject in need thereof.

In various embodiments, the inflammation is associated with a lung disease or disorder.

In various embodiments, the lung disease or disorder is selected from Asbestosis, Asthma, Bronchiectasis, Bronchitis, Chronic Cough, Chronic Obstructive Pulmonary Disease (COPD), Common Cold, Croup, Cystic Fibrosis, Hantavirus, Idiopathic Pulmonary Fibrosis, Influenza, Lung Cancer, Pandemic Flu, Pertussis, Pleurisy, Pneumonia, Pulmonary Embolism, Pulmonary Hypertension, Respiratory Syncytial Virus (RSV), Sarcoidosis, Sleep Apnea, Spirometry, Sudden Infant Death Syndrome (SIDS), and Tuberculosis.

In various embodiments, the inflammation is associated with a lung infection.

In various embodiments, the lung infection is cause by a bacterium, a fungus, a protozoa, a multi-cellular organism, a particulate, or a virus.

In various embodiments, the synthetic RNA is administered by inhalation.

In various embodiments, the inflammation is associated with sepsis.

In some aspects, the present invention relates to a method for treating alpha-1 antitrypsin (A1AT) deficiency. The method comprises a step of administering to a subject in need thereof an effective amount of (a) a synthetic RNA encoding a gene-editing protein capable of creating a double strand break in A1AT_A (SEQ ID NO: 584) and/or (b) a synthetic RNA encoding a gene-editing protein capable of creating a double strand break in A1AT_B (SEQ ID NO: 585), wherein the synthetic RNA comprises one or more non-canonical nucleotides that avoid substantial cellular toxicity. In various embodiments, the treatment comprises administering to a subject in need thereof an effective amount of (a) the synthetic RNA encoding the gene-editing protein capable of creating a double strand break in A1AT_A (SEQ ID NO: 584) and (b) the synthetic RNA encoding the a gene-editing protein capable of creating a double strand break in A1AT_B (SEQ ID NO: 585).

In various embodiments, the administration is directed to the liver.

In various embodiments, the administration is intraportal injection.

In various embodiments, the treatment corrects the Z mutation in the subject's liver cells.

In various embodiments, the treatment reduces polymerized Z protein accumulation in the subject's liver cells.

In various embodiments, the treatment increases secretion and/or serum levels of functional A1AT.

In various embodiments, the treatment ameliorates one or more of chronic cough, emphysema, COPD, liver failure, hepatitis, hepatomegaly, jaundice, and cirrhosis.

In various embodiments, the gene-editing protein is selected from a CRISPR/Cas9, TALEN, and a zinc finger nuclease.

In various embodiments, the gene-editing protein comprises: (a) a DNA-binding domain and (b) a nuclease domain. The DNA-binding domain comprises a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 629) and is between 36 and 39 amino acids long, wherein: “v” is Q, D or E; “w” is S or N; “x” is H, N, or I; “y” is D, A, I, N, G, H, K, S, or null; and “z” is GGKQALETVQRLLPVLCQD (SEQ ID NO: 630) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 631). The nuclease domain comprises a catalytic domain of a nuclease.

In various embodiments, the nuclease domain is capable of forming a dimer with another nuclease domain.

In various embodiments, the nuclease domain comprises the catalytic domain of a protein comprising the amino acid sequence of SEQ ID NO: 632.

In some aspects, the present invention relates to a method for treating alpha-1 antitrypsin (A1AT) deficiency. The method comprises a step of administering an effective amount of a synthetic RNA encoding A1AT to a subject, wherein the synthetic RNA comprises one or more non-canonical nucleotides that avoid substantial cellular toxicity.

In various embodiments, the administration is directed to the liver.

In various embodiments, the administration is intraportal injection.

In various embodiments, the treatment corrects the Z mutation in the subject's liver cells.

In various embodiments, the treatment reduces polymerized Z protein accumulation in the subject's liver cells.

In various embodiments, the treatment increases secretion and/or serum levels of functional A1AT.

In various embodiments, the treatment ameliorates one or more of the subject's symptoms.

In some aspects, the present invention relates to a method for reversing an alpha-1 antitrypsin (A1AT) deficiency in a cell. The method comprises steps of (a) obtaining a cell comprising a defective A1AT gene and (b) contacting the cell with a synthetic RNA encoding A1AT, wherein the synthetic RNA comprises one or more non-canonical nucleotides that avoid substantial cellular toxicity.

In various embodiments, the method further comprises steps of (c) obtaining the cell contacted in step (b); and (d) administering the cell to a subject in need thereof.

In various embodiments, the administration is directed to the liver.

In various embodiments, the administration is intraportal injection.

In some aspects, the present invention relates to a method for reversing an alpha-1 antitrypsin (A1AT) deficiency in a cell. The method comprises steps of (a) obtaining a cell comprising a defective A1AT gene and (b) contacting the cell with one or both of a synthetic RNA encoding a gene-editing protein capable of creating a double strand break in A1AT_A (SEQ ID NO: 584) and a synthetic RNA encoding a gene-editing protein capable of creating a double strand break in A1AT_B (SEQ ID NO: 585), wherein the synthetic RNA comprises one or more non-canonical nucleotides that avoid substantial cellular toxicity.

In various embodiments, the method further comprises steps of (c) obtaining the cell contacted in step (b); and (d) administering the cell to a subject in need thereof.

In various embodiments, the administration is directed to the liver.

In various embodiments, the administration is intraportal injection.

In various embodiments, the gene-editing protein is selected from a CRISPR/Cas9, TALEN and a zinc finger nuclease.

In various embodiments, the gene-editing protein comprises: (a) a DNA-binding domain and (b) a nuclease domain. The DNA-binding domain comprises a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 629) and is between 36 and 39 amino acids long, wherein: “v” is Q, D or E; “w” is S or N; “x” is H, N, or I; “y” is D, A, I, N, G, H, K, S, or null; and “z” is GGKQALETVQRLLPVLCQD (SEQ ID NO: 630) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 631). The nuclease domain comprises a catalytic domain of a nuclease.

In various embodiments, the nuclease domain is capable of forming a dimer with another nuclease domain.

In various embodiments, the nuclease domain comprises the catalytic domain of a protein comprising the amino acid sequence of SEQ ID NO: 632.

In some aspects, the present invention relates to a method for modulating BMP7. The method comprises a step of administering an effective amount of a synthetic RNA encoding BMP7 to a subject, wherein the synthetic RNA comprises one or more non-canonical nucleotides that avoid substantial cellular toxicity.

In various embodiments, the modulating results in an increase in the quantity of BMP7 in the subject.

In various embodiments, the modulating results in a decrease in the quantity of BMP7 in the subject.

In various embodiments, the modulating results in an increase in intracellular alkaline phosphatase activity.

In various embodiments, the synthetic RNA encoding BMP7 comprises a sequence encoding the BMP7 signal peptide.

In various embodiments, the BMP7 signal peptide comprises the sequence of SEQ ID NO: 597.

In various embodiments, the administration is directed to the liver.

In various embodiments, the administration is intraportal injection.

In various embodiments, the administration treats diabetic nephropathy.

In various embodiments, the administration treats liver fibrosis.

In various embodiments, the administration is directed to the kidney.

In various embodiments, the administration is intravenous or intradermal.

In various embodiments, the administration treats kidney disease.

In various embodiments, the kidney disease is diabetic nephropathy.

In various embodiments, the modulating results in reduced levels of urine albumin in the subject.

In some aspects, the present invention relates to a method for modulating an immune checkpoint molecule. The method comprises a step of administering an effective amount of a synthetic RNA encoding the immune checkpoint molecule to a subject, wherein the synthetic RNA comprises one or more non-canonical nucleotides that avoid substantial cellular toxicity.

In some aspects, the present invention relates to a method for treating a subject comprising administering a step of a synthetic RNA encoding a gene-editing protein targeting an immune checkpoint molecule gene.

In some aspects, the present invention relates to a method for treating a subject comprising steps of (a) obtaining a cell comprising an immune checkpoint molecule gene and (b) contacting the cell with a synthetic RNA encoding a gene-editing protein capable of creating a double strand break in the immune checkpoint molecule gene, wherein the synthetic RNA comprises one or more non-canonical nucleotides that avoid substantial cellular toxicity.

In various embodiments, the method further comprises steps of (c) obtaining the cell contacted in step (b); and (d) administering the cell to a subject in need thereof.

In various embodiments, the gene-editing protein is selected from a CRISPR/Cas9, TALEN and a zinc finger nuclease.

In various embodiments, the gene-editing protein comprises: (a) a DNA-binding domain and (b) a nuclease domain. The DNA-binding domain comprises a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 629) and is between 36 and 39 amino acids long, wherein:

“v” is Q, D or E; “w” is S or N; “x” is H, N, or I; “y” is D, A, I, N, G, H, K, S, or null; and “z” is GGKQALETVQRLLPVLCQD (SEQ ID NO: 630) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 631). The nuclease domain comprises a catalytic domain of a nuclease.

In various embodiments, the nuclease domain is capable of forming a dimer with another nuclease domain.

In various embodiments, the nuclease domain comprises the catalytic domain of a protein comprising the amino acid sequence of SEQ ID NO: 632.

In various embodiments, the immune checkpoint molecule is selected from PD-1, PD-L1, PD-L2, CTLA-4, ICOS, LAG3, OX40, OX40L, and TIM3.

In various embodiments, the immune checkpoint molecule is PD-1.

In various embodiments, the administering stimulates or enhances an immune response in the subject.

In various embodiments, the administering inhibits or reduces an immune response in the subject.

In various embodiments, the subject is afflicted with a cancer.

In various embodiments, the subject is afflicted with an autoimmune disease.

In various embodiments, the administration is intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intracerebral, intravaginal, transdermal, intraportal, rectally, by inhalation, or topically.

In various embodiments, the modulating results in an increase in the quantity of PD-1 in the subject.

In various embodiments, the modulating results in a decrease in the quantity of PD-1 in the subject.

In some aspects, the present invention relates to a method for treating a cancer comprising steps of (a) isolating an cell from a subject, the cell being an immune cell or hematopoietic cell; (b) contacting the isolated cell with an effective amount of a synthetic RNA encoding a chimeric antigen receptor (CAR), wherein the synthetic RNA comprises one or more non-canonical nucleotides that avoid substantial cellular toxicity; and (c) administering the cell to the subject.

In various embodiments, the immune cell is a T cell.

In some aspects, the present invention relates to a method for making a chimeric antigen receptor (CAR) T cell comprising steps of (a) obtaining a cell from a subject and (b) contacting the cell with a synthetic RNA encoding a gene-editing protein capable of creating a double strand break to yield a safe harbor locus for CAR insertion, wherein the synthetic RNA comprises one or more non-canonical nucleotides that avoid substantial cellular toxicity.

In various embodiments, the wherein the gene-editing protein is selected from a CRISPR/Cas9, TALEN and a zinc finger nuclease.

In various embodiments, the gene-editing protein comprises: (a) a DNA-binding domain and (b) a nuclease domain. The DNA-binding domain comprises a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 629) and is between 36 and 39 amino acids long, wherein: “v” is Q, D or E; “w” is S or N; “x” is H, N, or I; “y” is D, A, I, N, G, H, K, S, or null; and “z” is GGKQALETVQRLLPVLCQD (SEQ ID NO: 630) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 631). The nuclease domain comprises a catalytic domain of a nuclease.

In various embodiments, the nuclease domain is capable of forming a dimer with another nuclease domain.

In various embodiments, the nuclease domain comprises the catalytic domain of a protein comprising the amino acid sequence of SEQ ID NO: 632.

In some aspects, the present invention relates to a method for increasing the persistence of a chimeric antigen receptor (CAR) T cell, comprising a step of contacting the chimeric antigen receptor (CAR) T cell with a synthetic RNA encoding a encoding a telomerase, wherein the synthetic RNA comprises one or more non-canonical nucleotides that avoid substantial cellular toxicity.

In some aspects, the present invention relates to a method for treating a skin wound, comprising a step of administering an effective amount of a synthetic RNA encoding interleukin 22 (IL22) to a subject, wherein the synthetic RNA comprises one or more non-canonical nucleotides that avoid substantial cellular toxicity.

In various embodiments, the administration is directed to a population of cells of the integumentary system.

In various embodiments, the population of cells comprises one or more of cells of the epidermis, cells of the basement membrane, cells of the dermis, and/or cells of the subcutis.

In various embodiments, the population of cells is cells of the epidermis, and comprises one more of cells of the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and/or stratum germinativum.

In various embodiments, the population of cells is cells of the dermis, and comprises one or more of cells from the papillary region and the reticular region.

In various embodiments, the administration is by subcutaneous injection, intradermal injection, subdermal injection, intramuscular injection, or topical administration.

In various embodiments, the administration is intradermal injection to one or more of the dermis or epidermis.

In various embodiments, the an effective amount is from about 10 ng to about 5000 ng per treatment area of about 10 cm² or less, or about 5 cm² or less, or about 1 cm² or less, or about 0.5 cm² or less, or about 0.2 cm² or less.

In various embodiments, about 10 ng, or about 20 ng, or about 50 ng, or about 100 ng, or about 200 ng, or about 300 ng, or about 400 ng, or about 500 ng, or about 600 ng, or about 700 ng, or about 800 ng, or about 900 ng, or about 1000 ng, or about 1100 ng, or about 1200 ng, or about 1300 ng, or about 1400 ng, or about 1500 ng, or about 1600 ng, or about 1700 ng, or about 1800 ng, or about 1900 ng, or about 2000 ng, or about 3000 ng, or about 4000 ng, or about 5000 ng of the synthetic RNA is administered per treatment area of about 10 cm² or less, or about 5 cm² or less, or about 1 cm² or less, or about 0.5 cm² or less, or about 0.2 cm² or less.

In various embodiments, the modulating results in an increase in the quantity of IL22 in the subject.

In various embodiments, the modulating results in a decrease in the quantity of IL22 in the subject.

In various embodiments, the effective amount of synthetic RNA is administered using an array of needles covering an affected area of the subject.

In various embodiments, the treatment area is mechanically massaged after administration.

In various embodiments, the treatment area is exposed to electric pulses after administration.

In various embodiments, the electric pulses are between about 10V and about 200V for from about 50 microseconds to about 1 second.

In various embodiments, the electric pulses are generated around the treatment area by a multielectrode array.

In some aspects, the present invention relates to a method for treating a metabolic disorder, comprising a step of administering a synthetic RNA encoding a gene-editing protein targeting a defective gene, wherein the administration is by intraportal injection.

In some aspects, the present invention relates to a method for treating a metabolic disorder comprising a step of (a) obtaining a cell comprising a defective gene and (b) contacting the cell with a synthetic RNA encoding a gene-editing protein capable of creating a double strand break in the defective gene, wherein the synthetic RNA comprises one or more non-canonical nucleotides that avoid substantial cellular toxicity. In various embodiments, the method further comprises steps of (c) obtaining the cell contacted in step (b); and (d) administering the cell to a subject in need thereof by intraportal injection.

In various embodiments, the gene-editing protein is selected from a CRISPR/Cas9, TALEN and a zinc finger nuclease.

In various embodiments, the gene-editing protein comprises: (a) a DNA-binding domain and (b) a nuclease domain. The DNA-binding domain comprises a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 629) and is between 36 and 39 amino acids long, wherein: “v” is Q, D or E; “w” is S or N; “x” is H, N, or I; “y” is D, A, I, N, G, H, K, S, or null; and “z” is GGKQALETVQRLLPVLCQD (SEQ ID NO: 630) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 631). The nuclease domain comprises a catalytic domain of a nuclease.

In various embodiments, the nuclease domain is capable of forming a dimer with another nuclease domain.

In various embodiments, the nuclease domain comprises the catalytic domain of a protein comprising the amino acid sequence of SEQ ID NO: 632.

In various embodiments, the metabolic disorder is selected from a disorder of carbohydrate metabolism, a disorder of amino acid metabolism, a disorder of the urea cycle, a disorder of fatty acid metabolism, a disorder of porphyrin metabolism, a disorder of lysosomal storage, a disorder of peroxisome biogenesis, and a disorder of purine or pyrimidine metabolism.

In various embodiments, the metabolic disorder is a disorder of carbohydrate metabolism and wherein the disease is galactosemia and the defective gene is optionally GALT, GALK1, or GALE; wherein the disease is essential fructosuria and the defective gene is optionally KHK; wherein the disease is Hereditary fructose intolerance and the defective gene is optionally ALDOB; wherein the disease is glycogen storage disease type I and the defective gene is optionally G6PC, SLC37A4, or SLC17A3; wherein the disease is glycogen storage disease type II and the defective gene is optionally GAA; wherein the disease is glycogen storage disease type Ill and the defective gene is optionally AGL; wherein the disease is glycogen storage disease type IV and the defective gene is optionally GBE1; wherein the disease is glycogen storage disease type V and the defective gene is optionally PYGM; wherein the disease is glycogen storage disease type VI and the defective gene is optionally PYGL; wherein the disease is glycogen storage disease type VII and the defective gene is optionally PYGM; wherein the disease is glycogen storage disease type IX and the defective gene is optionally PHKA1, PHKA2, PHKB, PHKG1, or PHKG2; wherein the disease is glycogen storage disease type XI and the defective gene is optionally SLC2A2; wherein the disease is glycogen storage disease type XII and the defective gene is optionally ALDOA; wherein the disease is glycogen storage disease type XIII and the defective gene is optionally ENO1, ENO2, or ENO3; wherein the disease is glycogen storage disease type 0 and the defective gene is optionally GYS1 or GYS2; wherein the disease is pyruvate carboxylase deficiency and the defective gene is optionally PC; wherein the disease is pyruvate kinase deficiency and the defective gene is optionally PKLR; wherein the disease is transaldolase deficiency and the defective gene is optionally TALDO1; wherein the disease is triosephosphate isomerase deficiency and the defective gene is optionally TPI1; wherein the disease is fructose bisphosphatase deficiency and the defective gene is optionally FBP1; wherein the disease is hyperoxaluria and the defective gene is optionally AGXT or GRHPR; wherein the disease is hexokinase deficiency and the defective gene is optionally HK1; wherein the disease is glucose-galactose malabsorption and the defective gene is optionally SLC5A1; or wherein the disease is glucose-6-phosphate dehydrogenase deficiency and the defective gene is optionally G6PD.

In various embodiments, the metabolic disorder is a disorder of amino acid metabolism wherein the disease is alkaptonuria and the defective gene is optionally HGD; wherein the disease is aspartylglucosaminuria and the defective gene is optionally AGA; wherein the disease is methylmalonic acidemia and the defective gene is optionally MUT, MCEE, MMAA, MMAB, MMACHC, MMADHC, or LMBRD1; wherein the disease is maple syrup urine disease and the defective gene is optionally BCKDHA, BCKDHB, DBT, or DLD; wherein the disease is homocystinuria and the defective gene is optionally CBS; wherein the disease is tyrosinemia and the defective gene is optionally FAH, TAT, or HPD; wherein the disease is trimethylaminuria and the defective gene is optionally FMO3; wherein the disease is Hartnup disease and the defective gene is optionally SLC6A19; wherein the disease is biotinidase deficiency and the defective gene is optionally BTD; wherein the disease is ornithine carbamoyltransferase deficiency and the defective gene is optionally OTC; wherein the disease is carbamoyl-phosphate synthase I deficiency disease and the defective gene is optionally CPS1; wherein the disease is citrullinemia and the defective gene is optionally ASS or SLC25A13; wherein the disease is hyperargininemia and the defective gene is optionally ARG1; wherein the disease is hyperhomocysteinemia and the defective gene is optionally MTHFR; wherein the disease is hypermethioninemia and the defective gene is optionally MAT1A, GNMT, or AHCY; wherein the disease is hyperlysinemias and the defective gene is optionally AASS; wherein the disease is nonketotic hyperglycinemia and the defective gene is optionally GLDC, AMT, or GCSH; wherein the disease is Propionic acidemia and the defective gene is optionally PCCA or PCCB; wherein the disease is hyperprolinemia and the defective gene is optionally ALDH4A1 or PRODH; wherein the disease is cystinuria and the defective gene is optionally SLC3A1 or SLC7A9; wherein the disease is dicarboxylic aminoaciduria and the defective gene is optionally SLC1A1; wherein the disease is glutaric acidemia type 2 and the defective gene is optionally ETFA, ETFB, or ETFDH; wherein the disease is isovaleric acidemia and the defective gene is optionally IVD; or wherein the disease is 2-hydroxyglutaric aciduria and the defective gene is optionally L2HGDH or D2HGDH.

In various embodiments, the metabolic disorder is a disorder of the urea cycle wherein the disease is N-acetylglutamate synthase deficiency and the defective gene is optionally NAGS; wherein the disease is argininosuccinic aciduria and the defective gene is optionally ASL; or wherein the disease is argininemia and the defective gene is optionally ARG1.

In various embodiments, the metabolic disorder is a disorder of fatty acid metabolism wherein the disease is very long-chain acyl-coenzyme A dehydrogenase deficiency and the defective gene is optionally ACADVL; wherein the disease is long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency and the defective gene is optionally HADHA; wherein the disease is medium-chain acyl-coenzyme A dehydrogenase deficiency and the defective gene is optionally ACADM; wherein the disease is short-chain acyl-coenzyme A dehydrogenase deficiency and the defective gene is optionally ACADS; wherein the disease is 3-hydroxyacyl-coenzyme A dehydrogenase deficiency and the defective gene is optionally HADH; wherein the disease is 2,4 dienoyl-CoA reductase deficiency and the defective gene is optionally NADK2; wherein the disease is 3-hydroxy-3-methylglutaryl-CoA lyase deficiency and the defective gene is optionally HMGCL; wherein the disease is malonyl-CoA decarboxylase deficiency and the defective gene is optionally MLYCD; wherein the disease is systemic primary carnitine deficiency and the defective gene is optionally SLC22A5; wherein the disease is carnitine-acylcarnitine translocase deficiency and the defective gene is optionally SLC25A20; wherein the disease is carnitine palmitoyltransferase I deficiency and the defective gene is optionally CPT1A; wherein the disease is carnitine palmitoyltransferase II deficiency and the defective gene is optionally CPT2; wherein the disease is lysosomal acid lipase deficiency and the defective gene is optionally LIPA; or wherein the disease is Gaucher's disease and the defective gene is optionally GBA.

In various embodiments, the metabolic disorder is a disorder of porphyrin metabolism wherein the disease is acute intermittent porphyria and the defective gene is optionally HMBS; wherein the disease is Gunther disease and the defective gene is optionally UROS; wherein the disease is porphyria cutanea tarda and the defective gene is optionally UROD; wherein the disease is hepatoerythropoietic porphyria and the defective gene is optionally UROD; wherein the disease is hereditary coproporphyria and the defective gene is optionally CPOX; wherein the disease is variegate porphyria and the defective gene is optionally PPOX; wherein the disease is erythropoietic protoporphyria and the defective gene is optionally FECH; or wherein the disease is aminolevulinic acid dehydratase deficiency porphyria and the defective gene is optionally ALAD.

In various embodiments, the metabolic disorder is a disorder of lysosomal storage wherein the disease is Farber disease and the defective gene is optionally ASAH1; wherein the disease is Krabbe disease and the defective gene is optionally GALC; wherein the disease is galactosialidosis and the defective gene is optionally CTSA; wherein the disease is fabry disease and the defective gene is optionally GLA; wherein the disease is Schindler disease and the defective gene is optionally NAGA; wherein the disease is GM1 gangliosidosis and the defective gene is optionally GLB1; wherein the disease is Tay-Sachs disease and the defective gene is optionally HEXA; wherein the disease is Sandhoff disease and the defective gene is optionally HEXB; wherein the disease is GM2-gangliosidosis, AB variant and the defective gene is optionally GM2A; wherein the disease is Niemann-Pick disease and the defective gene is optionally SMPD1, NPC1, or NPC2; wherein the disease is metachromatic leukodystrophy and the defective gene is optionally ARSA or PSAP; wherein the disease is multiple sulfatase deficiency and the defective gene is optionally SUMF1; wherein the disease is Hurler syndrome and the defective gene is optionally IDUA; wherein the disease is Hunter syndrome and the defective gene is optionally IDS; wherein the disease is Sanfilippo syndrome and the defective gene is optionally SGSH, NAGLU, HGSNAT, or GNS; wherein the disease is Morquio syndrome and the defective gene is optionally GALNS or GLB1; wherein the disease is Maroteaux-Lamy syndrome and the defective gene is optionally ARSB; wherein the disease is Sly syndrome and the defective gene is optionally GUSB; wherein the disease is sialidosis and the defective gene is optionally NEU1, NEU2, NEU3, or NEU4; wherein the disease is I-cell disease and the defective gene is optionally GNPTAB or GNPTG; wherein the disease is mucolipidosis type IV and the defective gene is optionally MCOLN1; wherein the disease is infantile neuronal ceroid lipofuscinosis and the defective gene is optionally PPT1 or PPT2; wherein the disease is Jansky-Bielschowsky disease and the defective gene is optionally TPP1; wherein the disease is Batten disease and the defective gene is optionally CLN1, CLN2, CLN3, CLN5, CLN6, MFSD8, CLN8, or CTSD; wherein the disease is Kufs disease, Type A and the defective gene is optionally CLN6 or PPT1; wherein the disease is Kufs disease, Type B and the defective gene is optionally DNAJC5 or CTSF; wherein the disease is alpha-mannosidosis and the defective gene is optionally MAN2B1, MAN2B2, or MAN2C1; wherein the disease is beta-mannosidosis and the defective gene is optionally MANBA; wherein the disease is fucosidosis and the defective gene is optionally FUCA1; wherein the disease is cystinosis and the defective gene is optionally CTNS; wherein the disease is pycnodysostosis and the defective gene is optionally CTSK; wherein the disease is Salla disease and the defective gene is optionally SLC17A5; wherein the disease is Infantile free sialic acid storage disease and the defective gene is optionally SLC17A5; or wherein the disease is Danon disease and the defective gene is optionally LAMP2.

In various embodiments, the metabolic disorder is a disorder of peroxisome biogenesis wherein the disease is Zellweger syndrome and the defective gene is optionally PEX1, PEX2, PEX3, PEX5, PEX6, PEX12, PEX14, or PEX26; wherein the disease is Infantile Refsum disease and the defective gene is optionally PEX1, PEX2, or PEX26; wherein the disease is neonatal adrenoleukodystrophy and the defective gene is optionally PEX5, PEX1, PEX10, PEX13, or PEX26; wherein the disease is RCDP Type 1 and the defective gene is optionally PEX7; wherein the disease is pipecolic acidemia and the defective gene is optionally PAHX; wherein the disease is acatalasia and the defective gene is optionally CAT; wherein the disease is hyperoxaluria type 1 and the defective gene is optionally AGXT; wherein the disease is Acyl-CoA oxidase deficiency and the defective gene is optionally ACOX1; wherein the disease is D-bifunctional protein deficiency and the defective gene is optionally HSD17B4; wherein the disease is dihydroxyacetonephosphate acyltransferase deficiency and the defective gene is optionally GNPAT; wherein the disease is X-linked adrenoleukodystrophy and the defective gene is optionally ABCD1; wherein the disease is α-methylacyl-CoA racemase deficiency and the defective gene is optionally AMACR; wherein the disease is RCDP Type 2 and the defective gene is optionally DHAPAT; wherein the disease is RCDP Type 3 and the defective gene is optionally AGPS; wherein the disease is adult refsum disease-1 and the defective gene is optionally PHYH; or wherein the disease is mulibrey nanism and the defective gene is optionally TRIM37.

In various embodiments, the metabolic disorder is a disorder of purine or pyrimidine metabolism wherein the disease is Lesch-Nyhan syndrome and the defective gene is optionally HPRT; wherein the disease is adenine phosphoribosyltransferase deficiency and the defective gene is optionally APRT; wherein the disease is adenosine deaminase deficiency and the defective gene is optionally ADA; wherein the disease is Adenosine monophosphate deaminase deficiency type 1 and the defective gene is optionally AMPD1; wherein the disease is adenylosuccinate lyase deficiency and the defective gene is optionally ADSL; wherein the disease is dihydropyrimidine dehydrogenase deficiency and the defective gene is optionally DPYD; wherein the disease is Miller syndrome and the defective gene is optionally DHODH; wherein the disease is orotic aciduria and the defective gene is optionally UMPS; wherein the disease is purine nucleoside phosphorylase deficiency and the defective gene is optionally PNP; or wherein the disease is xanthinuria and the defective gene is optionally XDH, MOCS1, or MOCS2, GEPH.

In some aspects, the present invention relates to a method for modulating transthyretin (TTR). The method comprising a step of administering an effective amount of a synthetic RNA encoding TTR to a subject, wherein the synthetic RNA comprises one or more non-canonical nucleotides that avoid substantial cellular toxicity.

In various embodiments, the modulating results in an increase in the quantity of TTR in the subject.

In various embodiments, the modulating results in a decrease in the quantity of TTR in the subject.

In various embodiments, the modulating results in treatment of one or more of an amyloid disease, senile systemic amyloidosis (SSA), familial amyloid polyneuropathy (FAP), and familial amyloid cardiomyopathy (FAC).

In various embodiments, the non-canonical nucleotides have one or more substitutions at positions selected from the 2C, 4C, and 5C positions for a pyrimidine, or selected from the 6C, 7N and 8C positions for a purine.

In various embodiments, the non-canonical nucleotides comprise one or more of 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine, optionally at an amount of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or 100% of the non-canonical nucleotides.

In various embodiments, the at least about 50% of cytidine residues are non-canonical nucleotides, and which are selected from 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, and 5-methoxycytidine.

In various embodiments, the at least about 75% or at least about 90% of cytidine residues are non-canonical nucleotides, and the non-canonical nucleotides are selected from 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, and 5-methoxycytidine.

In various embodiments, the at least about 20% of uridine, or at least about 40%, or at least about 50%, or at least about 75%, or at about least 90% of uridine residues are non-canonical nucleotides, and the non-canonical are selected from pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine.

In various embodiments, the at least about 40%, or at least about 50%, or at least about 75%, or at about least 90% of uridine residues are non-canonical nucleotides, and the non-canonical nucleotides are selected from pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine.

In various embodiments, the at least about 10% of guanine residues are non-canonical nucleotides, and the non-canonical nucleotide is optionally 7-deazaguanosine.

In various embodiments, the synthetic RNA contains no more than about 50% 7-deazaguanosine in place of guanosine residues.

In various embodiments, the synthetic RNA does not contain non-canonical nucleotides in place of adenosine residues.

In various embodiments, the synthetic RNA comprises a 5′ cap structure.

In various embodiments, the synthetic RNA 5′-UTR comprises a Kozak consensus sequence.

In various embodiments, the synthetic RNA 5′-UTR comprises a sequence that increases RNA stability in vivo, and the 5′-UTR may comprise an alpha-globin or beta-globin 5′-UTR.

In various embodiments, the synthetic RNA 3′-UTR comprises a sequence that increases RNA stability in vivo, and the 3′-UTR may comprise an alpha-globin or beta-globin 3′-UTR.

In various embodiments, the synthetic RNA comprises a 3′ poly(A) tail.

In various embodiments, the synthetic RNA 3′ poly(A) tail is from about 20 nucleotides to about 250 nucleotides in length.

In various embodiments, the synthetic RNA is from about 200 nucleotides to about 5000 nucleotides in length.

In various embodiments, the synthetic RNA is from about 500 to about 2000 nucleotides in length, or about 500 to about 1500 nucleotides in length, or about 500 to about 1000 nucleotides in length.

In various embodiments, the synthetic RNA is prepared by in vitro transcription.

In various embodiments, the effective amount of the synthetic RNA is administered as one or more injections containing about 10 ng to about 5000 ng of RNA.

In various embodiments, the effective amount of the synthetic RNA is administered as one or more injections each containing no more than about 10 ng, or no more than about 20 ng, or no more than about 50 ng, or no more than about 100 ng, or no more than about 200 ng, or no more than about 300 ng, or no more than about 400 ng, or no more than about 500 ng, or no more than about 600 ng, or no more than about 700 ng, or no more than about 800 ng, or no more than about 900 ng, or no more than about 1000 ng, or no more than about 1100 ng, or no more than about 1200 ng, or no more than about 1300 ng, or no more than about 1400 ng, or no more than about 1500 ng, or no more than about 1600 ng, or no more than about 1700 ng, or no more than about 1800 ng, or no more than about 1900 ng, or no more than about 2000 ng, or no more than about 3000 ng, or no more than about 4000 ng, or no more than about 5000 ng.

In various embodiments, the effective amount of the synthetic RNA is administered as one or more injections each containing about 10 ng, or about 20 ng, or about 50 ng, or about 100 ng, or about 200 ng, or about 300 ng, or about 400 ng, or about 500 ng, or about 600 ng, or about 700 ng, or about 800 ng, or about 900 ng, or about 1000 ng, or about 1100 ng, or about 1200 ng, or about 1300 ng, or about 1400 ng, or about 1500 ng, or about 1600 ng, or about 1700 ng, or about 1800 ng, or about 1900 ng, or about 2000 ng, or about 3000 ng, or about 4000 ng, or about 5000 ng.

In various embodiments, the effective amount of the synthetic RNA comprises one or more lipids to enhance uptake of RNA by cells.

In various embodiments, the effective amount of the synthetic RNA comprises a cationic liposome formulation and the lipids are optionally selected from Table 1.

In various embodiments, the subject is a human.

In various embodiments, the effective amount of the synthetic RNA is administered about weekly, for at least 2 weeks.

In various embodiments, the effective amount of the synthetic RNA is administered about every other week for at least one month.

In various embodiments, the effective amount of the synthetic RNA is administered monthly or about every other month.

In various embodiments, the effective amount of the synthetic RNA is administered for at least two months, or at least 4 months, or at least 6 months, or at least 9 months, or at least one year.

In some aspects, the present invention relates to a composition comprising an effective amount of the synthetic RNA used in any herein-disclosed aspect or embodiment.

In some aspects, the present invention relates to a pharmaceutical composition, comprising the composition of any herein-disclosed aspect or embodiment and a pharmaceutically acceptable excipient.

In some aspects, the present invention relates to the use of a composition or pharmaceutical composition of any herein-disclosed aspect or embodiment in the treatment of a disease or disorder described herein.

In some aspects, the present invention relates to the use of a composition or pharmaceutical composition of any herein-disclosed aspect or embodiment in the manufacture of a medicament for the treatment of a disease or disorder described herein.

In various aspects, the present invention is based on the surprising discovery of safe and effective doses and administration parameters for nucleic acid drugs in human subjects. In some aspects, there is provided a method for delivering a nucleic acid drug, comprising administering an effective dose of a nucleic acid drug to a human subject in need thereof. In various embodiments, the nucleic acid drug comprises a RNA comprising one or more non-canonical nucleotides (a/k/a “modified RNA”). In other embodiments, the effective dose is an amount sufficient to substantially increase an amount of a protein encoded by the nucleic acid drug in the human subject and/or substantially avoid an immune reaction in a human subject, wherein the immune reaction is optionally mediated by the innate immune system.

In some aspects, there is provided a method for expressing a protein of interest in a population of cells in a mammalian subject, comprising administering a non-viral transfection composition comprising an effective dose of a RNA encoding the protein of interest to said cells, where the transfection composition is administered in an amount that allows for expression of said protein in said cells for at least about six hours to about five days without substantial cellular toxicity. In some embodiments, the RNA contains one or more non-canonical nucleotides that avoid substantial cellular toxicity.

In some embodiments, the effective dose is about 100 ng to about 5000 ng (e.g., about, or no more than about, 100 ng, or 200 ng, or 300 ng, or 400 ng, or 500 ng, or 600 ng, or 700 ng, or 800 ng, or 900 ng, or 1000 ng, or 1100 ng, or 1200 ng, or 1300 ng, or 1400 ng, or 1500 ng, or 1600 ng, or 1700 ng, or 1800 ng, or 1900 ng, or 2000 ng, or 3000 ng, or 4000 ng, or 5000 ng). In other embodiments, the effective dose is less than about 100 ng. In certain embodiments, the effective dose is about 10 ng to about 100 ng (e.g., about, or no more than about, 10 ng, or 20 ng, or 30 ng, or 40 ng, or 50 ng, or 60 ng, or 70 ng, or 80 ng, or 90 ng, or 100 ng).

In some embodiments, the effective dose is about 1.4 ng/kg to about 30 ng/kg (e.g., about, or no more than about, 1.4 ng/kg, or 2.5 ng/kg, or 5 ng/kg, or 10 ng/kg, or 15 ng/kg, or 20 ng/kg, or 25 ng/kg, or 30 ng/kg. In other embodiments, the effective dose is less than about 1.5 ng/kg. In certain embodiments, the effective dose is about 0.14 ng/kg to about 1.4 ng/kg (e.g., about, or no more than about, 0.14 ng/kg, or 0.25 ng/kg, or 0.5 ng/kg, or 0.75 ng/kg, or 1 ng/kg, or 1.25 ng/kg, or 1.4 ng/kg).

In some embodiments, the effective dose is about 350 ng/cm² to about 7000 ng/cm² (e.g., about, or no more than about, 350 ng/cm², or 500 ng/cm², or 750 ng/cm², or 1000 ng/cm², or 2000 ng/cm², or 3000 ng/cm², or 4000 ng/cm², or 5000 ng/cm², or 6000 ng/cm², or 7000 ng/cm²). In other embodiments, the effective dose is less than about 350 ng/cm². In certain embodiments, the effective dose is about 35 ng/cm² to about 350 ng/cm² (e.g., about, or no more than about, 35 ng/cm², or 50 ng/cm², or 75 ng/cm², or 100 ng/cm², or 150 ng/cm², or 200 ng/cm², or 250 ng/cm², or 300 ng/cm², or 350 ng/cm².

In some embodiments, the effective dose is about 0.28 picomoles to about 5.7 picomoles (e.g., about, or no more than about, 0.28 picomoles, or 0.5 picomoles, or 0.75 picomoles, or 1 picomole, or 2 picomoles, or 3 picomoles, or 4 picomoles, or 5 picomoles, or 5.7 picomoles). In other embodiments, the effective dose is less than about 0.28 picomoles. In certain embodiments, the effective dose is about 0.028 picomoles to about 0.28 picomoles (e.g., about, or no more than about, 0.028 picomoles, or 0.05 picomoles, or 0.075 picomoles, or 0.1 picomoles, or 0.15 picomoles, or 0.2 picomoles, or 0.25 picomoles, or 0.28 picomoles).

In some embodiments, the effective dose is about 0.004 picomoles/kg to about 0.082 picomoles/kg (e.g., about, or no more than about, 0.004 picomoles/kg, or 0.01 picomoles/kg, or 0.02 picomoles/kg, or 0.03 picomoles/kg, or 0.04 picomoles/kg, or 0.05 picomoles/kg, or 0.06 picomoles/kg, or 0.07 picomoles/kg, or 0.08 picomoles/kg, or 0.082 picomoles/kg). In other embodiments, the effective dose is less than about 0.004 picomoles/kg. In certain embodiments, the effective dose is about 0.0004 picomoles/kg to about 0.004 picomoles/kg (e.g., about, or no more than about, 0.0004 picomoles/kg, or 0.001 picomoles/kg, or 0.002 picomoles/kg, or 0.003 picomoles/kg, or 0.004 picomoles/kg).

In some embodiments, the effective dose is about 1 picomole/cm² to about 20 picomoles/cm² (e.g., about, or no more than about, 1 picomole/cm², or 2 picomoles/cm², or 3 picomoles/cm², or 4 picomoles/cm², or 5 picomoles/cm², or 6 picomoles/cm², or 7 picomoles/cm², or 8 picomoles/cm², or 9 picomoles/cm², or 10 picomoles/cm², or 12 picomoles/cm², or 14 picomoles/cm², or 16 picomoles/cm², or 18 picomoles/cm², or 20 picomoles/cm²). In other embodiments, the effective dose is less than about 1 picomole/cm². In certain embodiments, the effective dose is about 0.1 picomoles/cm² to about 1 picomole/cm² (e.g., about, or no more than about, 0.1 picomoles/cm², or 0.2 picomoles/cm², or 0.3 picomoles/cm², or 0.4 picomoles/cm², or 0.5 picomoles/cm², or 0.6 picomoles/cm², or 0.7 picomoles/cm², or 0.8 picomoles/cm², or 0.9 picomoles/cm², or 1 picomole/cm²).

In various embodiments, the nucleic acid drug is administered in a pharmaceutically acceptable formulation, including a formulation suitable for one or more of injection (e.g. subcutaneous injection, intradermal injection (including to the dermis or epidermis), subdermal injection, intramuscular injection, intraocular injection, intravitreal injection, intra-articular injection, intracardiac injection, intravenous injection, epidural injection, intrathecal injection, intraportal injection, intratumoral injection) and topical administration and/or for administration to the integumentary system (e.g. to one or more of the epidermis (optionally selected from the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum germinativum), basement membrane, dermis (optionally selected from the papillary region and the reticular region), subcutis, and conjunctiva) and/or for administration to the eye (e.g., to one or more of the cornea, sclera, iris, lens, corneal limbus, optic nerve, choroid, ciliary body, anterior segment, anterior chamber, and retina).

In various embodiments, the nucleic acid drug is formulated with one or more lipids to enhance uptake of the nucleic acid drug by cells, the lipids optionally selected from Table 1. In other embodiments, the nucleic acid drug is formulated with one or more nanoparticles, optionally lipid or polymeric nanoparticles, to enhance uptake of the nucleic acid drug by cells, to enhance duration of protein expression, or to otherwise enhance the safety and/or efficacy of the nucleic acid drug.

In various embodiments, the nucleic acid drug is administered locally, optionally by one or more of subcutaneous injection, intradermal injection, subdermal injection and intramuscular injection, and the effective dose is administered to a surface area of about 4 mm² to about 1000 mm² (e.g. about, or no more than about, 4 mm², or 5 mm², or 10 mm², or 25 mm², or 50 mm², or 75 mm², or 100 mm², or 125 mm², or 150 mm², or 200 mm², or 500 mm², or 1000 mm²).

In various embodiments, the nucleic acid drug is administered in a treatment regimen, optionally with an additional agent or adjuvant therapy described herein, and the administration is about weekly to about once every 24 weeks (e.g. about, or not more than about, weekly, or once every 2 weeks, or once every 3 weeks, or once every 4 weeks, or once every 5 weeks, or once every 6 weeks, or once every 7 weeks, or once every 8 weeks, or once every 9 weeks, or once every 9 weeks, or once every 9 weeks, or once every 9 weeks, or once every 10 weeks, or once every 11 weeks, or once every 12 weeks, or once every 13 weeks, or once every 14 weeks, or once every 15 weeks, or once every 20 weeks, or once every 24 weeks). In other embodiments, the nucleic acid drug is administered in a treatment regimen, optionally with an additional agent or adjuvant therapy described herein, and the administration is about daily to about weekly (e.g., about, or not more than about, daily, or once every 2 days, or once every 3 days, or once every 4 days, or once every 5 days, or once every 6 days, or weekly).

In various embodiments the nucleic acid drug comprises RNA comprising one or more non-canonical nucleotides, optionally having one or more substitutions at the 2C and/or 4C and/or 5C positions for a pyrimidine or the 6C and/or 7N and/or 8C positions for a purine. In various embodiments, the non-canonical nucleotide is one or more of the non-canonical nucleotides described herein, including, for example, 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, pseudouridine, 5-hydroxyuridine, 5-hydroxypseudouridine, 5-methyluridine, 5-methylpseudouridine, 5-hydroxymethyluridine, 5-hydroxymethylpseudouridine, 5-carboxyuridine, 5-carboxypseudouridine, 5-formyluridine, 5-formylpseudouridine, 5-methoxyuridine, and 5-methoxypseudouridine. Further, the RNA comprising one or more non-canonical nucleotides may have one or more of a 5′-UTR comprising a Kozak consensus sequence, a 5′-UTR or 3′-UTR comprising a sequence that increases RNA stability in vivo (e.g. an alpha-globin or beta-globin 5′-UTR or an alpha-globin or beta-globin 3′-UTR), and a 3′ poly(A) tail from about 20 nucleotides to about 250 nucleotides in length (e.g. about 20, or about 30, or about 40, or about 50, or about 60, or about 70, or about 80, or about 90, or about 100, or about 110, or about 120, or about 130, or about 140, or about 150, or about 160, or about 170, or about 180, or about 190, or about 200, or about 210, or about 220, or about 230, or about 240, or about 250 nucleotides in length).

Further, some aspects of the methods described herein find use in various medical treatments, including, by way of illustration, treating a disease, disorder and/or condition of the integumentary system or altering, modifying and/or changing the integumentary system (e.g. cosmetically).

Also contemplated are kits suitable for use in human therapy, comprising the nucleic acid drug described herein in a unit dosage form of about 10 ng to about 5000 ng (e.g. about, or no more than about, 10 ng, or 20 ng, or 50 ng, or 100 ng, or 200 ng, or 300 ng, or 400 ng, or 500 ng, or 600 ng, or 700 ng, or 800 ng, or 900 ng, or 1000 ng, or 1100 ng, or 1200 ng, or 1300 ng, or 1400 ng, or 1500 ng, or 1600 ng, or 1700 ng, or 1800 ng, or 1900 ng, or 2000 ng, or 3000 ng, or 4000 ng, or 5000 ng) and an injection needle.

Further, in some aspects, the present invention provides a pharmaceutical formulation comprising the nucleic acid drug described herein and one or more of the vehicles (a/k/a “transfection reagents”, e.g., lipids) described herein, the formulation optionally being suitable for one or more of subcutaneous injection, intradermal injection, subdermal injection, intramuscular injection, intraocular injection, intravitreal injection, intra-articular injection, intracardiac injection, intravenous injection, epidural injection, intrathecal injection, intraportal injection, intratumoral injection, and topical administration. As used herein, the term “injection” can refer to injection, for example, with a syringe, and to other methods of administering liquids, for example, infusion, perfusion, administration using a pen injector, cartridge system needle-array or patch and/or administration by a catheter system.

In some aspects, nucleic acid delivery patches are provided. In one aspect, devices for delivering nucleic acids using electric fields are provided. Other aspects pertain to methods and compositions for delivery of nucleic acids to the skin. Still further aspects pertain to methods and compositions for expression of proteins in the skin.

In one aspect, the invention provides methods and compositions for treating diseases and conditions in humans, including, but not limited to, prophylactic treatments, treatments for rare diseases, including, but not limited to, dermatologic rare diseases, and treatments for use in medical dermatology and aesthetic medicine. In another aspect, the invention provides cosmetics comprising nucleic acids. Still further aspects relate to methods and compositions for altering pigmentation, for example, for the treatment of pigmentation disorders. Still further aspects relate to methods and compositions for enhancing healing, including, but not limited to, healing in response to a wound or surgery. The compositions of the present invention may alter, modify and/or change the appearance of a member of the integumentary system of a subject such as, but not limited, to skin, hair and nails. Such alteration, modification and/or change may be in the context of treatment methods and/or therapeutic uses as described herein including, by way of non-limiting example, dermatological treatments and cosmetics procedures.

Further, in various embodiments, the present invention relates to the targeting of various therapeutic proteins that are not limited to dermatological applications. For example, in various embodiments, the present compositions and methods find use in methods of treatment that are mediated by the increased expression of, for example, various soluble proteins as illustrated herein. In various embodiments, the nucleic acid drug encodes and/or increases the expression of one or more of a circulating protein, an extracellular matrix protein, an engineered protein, a gene-editing protein, a protein or peptide hormone, an enzyme, erythropoietin, darbepoetin alfa, NOVEPOETIN, elastin, collagen, an antibody or antibody fragment (e.g., a neutralizing antibody or antibody fragment), an intracellular protein, telomerase reverse transcriptase, a membrane protein, a fusion protein, a receptor, a ligand binding domain, a protein inhibitor, or a biologically active fragment, analogue or variant thereof. In other embodiments, administration of the nucleic acid drug results in one or more of an increase in hematocrit, an increase in tissue elasticity, an increase in tissue strength, and increase in skin hydration and/or water retention, hair growth, fat reduction, an insertion, deletion or mutation in DNA, conversion of a prodrug to an active drug, a decrease in tumor size and/or number, a decrease in plaque size and/or number, an increase in vascularization, a decrease in vascularization, an increase in visual acuity, a decrease in pain, an increase in cardiac output (e.g., ejection fraction and stroke volume), a decrease in abnormal heart rhythm, a decrease in fibrosis, a decrease in one or more adverse neurological symptoms, conditions, or disorders (e.g., depression, dysregulation of appetite, polyphagia, anorexia, dementia, headache, fatigue, numbness, tremors, and dizziness), a decrease in erectile dysfunction, an increase in vitality, an increase in pulmonary function, an increase in kidney function, an increase in liver function, an increase in insulin sensitivity, a decrease in insulin sensitivity, a decrease in inflammation, an increase in tear production, an improvement in hearing, an increase in auditory perception, a decrease in tinnitus, a reduction in perspiration, partial or total clearance of an infection, an increase in fertility, a decrease in fertility, inhibition or neutralization of a protein, recruitment or stimulation of one or more components of the immune system, lengthening of telomeres, inhibition of cellular senescence, an increase in replicative potential, reprogramming, proliferation, differentiation, and an increase in differentiation potential.

In some aspects, RNA molecules with low toxicity and high translation efficiency are provided. In one aspect, a cell-culture medium for high-efficiency in vivo transfection, reprogramming, and gene editing of cells is provided. Other aspects pertain to methods for producing RNA molecules encoding reprogramming proteins. Still further aspects pertain to methods for producing RNA molecules encoding gene-editing proteins.

In one aspect, the invention provides high-efficiency gene-editing proteins comprising engineered nuclease cleavage domains. In another aspect, the invention provides high-fidelity gene-editing proteins comprising engineered nuclease cleavage domains. Other aspects relate to high-efficiency gene-editing proteins comprising engineered DNA-binding domains. Still further aspects pertain to high-fidelity gene-editing proteins comprising engineered DNA-binding domains. Still further aspects relate to gene-editing proteins comprising engineered repeat sequences. Still further aspects relate to gene-editing proteins comprising DNA-modification domains. Some aspects relate to methods for altering the DNA sequence of a cell by transfecting the cell with or inducing the cell to express a gene-editing protein. Other aspects relate to methods for altering the DNA sequence of a cell that is present in an in vitro culture. Still further aspects relate to methods for altering the DNA sequence of a cell that is present in vivo.

In some aspects, the invention provides methods for treating cancer comprising administering to a patient a therapeutically effective amount of a gene-editing protein or a nucleic-acid encoding a gene-editing protein. In one aspect, the gene-editing protein is capable of altering the DNA sequence of a cancer associated gene. In another aspect, the cancer-associated gene is the BIRC5 gene. In other aspects, the gene-editing protein is capable of altering the DNA sequence of a cell to cause the cell to express an antigen or antigen receptor. In one aspect, the antigen is a tumor antigen. In another aspect, the antigen receptor binds to a tumor antigen. In yet another aspect, the antigen receptor is a chimeric antigen receptor. Still other aspects relate to therapeutics comprising nucleic acids and/or cells and methods of using therapeutics comprising nucleic acids and/or cells for the treatment of, for example, type 1 diabetes, heart disease, including ischemic and dilated cardiomyopathy, macular degeneration, Parkinson's disease, cystic fibrosis, sickle-cell anemia, thalassemia, Fanconi anemia, severe combined immunodeficiency, hereditary sensory neuropathy, xeroderma pigmentosum, Huntington's disease, muscular dystrophy, amyotrophic lateral sclerosis, Alzheimer's disease, cancer, and infectious diseases including hepatitis and HIV/AIDS. In some aspects, the nucleic acids comprise RNA. In other aspects, the RNA comprises one or more non-canonical nucleotides. In still other aspects, the nucleic acids are delivered to cells using a virus. In some aspects, the virus is a replication-competent virus. In other aspects, the virus is a replication-incompetent virus.

The details of the invention are set forth in the accompanying description below. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, illustrative methods and materials are now described. Other features, objects, and advantages of the invention will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Any aspect or embodiment disclosed herein can be combined with any other aspect or embodiment as disclosed herein.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1 depicts primary adult human dermal fibroblasts transfected with RNA encoding green fluorescent protein (“GFP”) and comprising the indicated nucleotides.

FIG. 2 depicts the result of a gene-expression analysis of the primary adult human dermal fibroblasts of FIG. 1 using a one-step real-time RT-PCR and primers designed to amplify human interferon beta mRNA. Data were normalized to the untransfected sample (“Neg.”). GAPDH was used as a loading control.

FIG. 3 depicts the results of a gene-expression analysis of cells transfected with RNA comprising the indicated nucleotides, conducted as in FIG. 2. Data were normalized to the untransfected sample (“Neg.”). GAPDH was used as a loading control.

FIG. 4 depicts the results of a gene-expression analysis of cells transfected with RNA comprising the indicated nucleotides, conducted as in FIG. 2, and using primers designed to amplify the indicated transcripts. Data were normalized to the untransfected sample (“Neg.”). GAPDH was used as a loading control.

FIG. 5 depicts the results of a gene-expression analysis of primary human epidermal keratinocytes transfected with RNA encoding NOVEPOETIN, and comprising the indicated nucleotides, conducted as in FIG. 2. Data were normalized to the untransfected sample (“Neg.”). GAPDH was used as a loading control.

FIG. 6 depicts intradermal injection of a solution comprising RNA encoding GFP into the ventral forearm of a healthy, 33 year-old, 70 kg, male human subject.

FIG. 7 depicts a region of the ventral forearm of the subject shown in FIG. 6 after treatment with RNA comprising 5-methoxyuridine and encoding GFP (injection sites 1-3) or COL7 (injection site 4). The image was taken immediately following the final injection.

FIG. 8 depicts the region of FIG. 7, 24 hours after injection.

FIG. 9 depicts the results of fluorescent imaging of the region of FIG. 7, using the indicated fluorescent channels. The dose at each injection site is also indicated. Images were taken 24 hours after injection.

FIG. 10 depicts the results of fluorescent imaging of the region of FIG. 7, using the FITC fluorescent channel. The dose at each injection site is indicated. Images were taken 48 hours after injection.

FIG. 11 depicts the results of quantitative fluorescent imaging of the region of FIG. 7, using the FITC fluorescent channel. The horizontal axis indicates time after injection.

FIG. 12 depicts the results of fluorescent imaging of an independent experiment in which a region of the ventral forearm of as the subject shown in FIG. 6 treated with RNA comprising 5-methoxyuridine and encoding GFP. The image was taken 24 hours after injection.

FIG. 13 depicts the results of an ELISA designed to detect darbepoetin alfa in culture media of primary human epidermal keratinocytes transfected with RNA comprising the indicated nucleotides and encoding NOVEPOETIN.

FIG. 14 depicts the results of an ELISA designed to detect darbepoetin alfa in culture media of primary human epidermal keratinocytes transfected with RNA comprising the indicated nucleotides and encoding NOVEPOETIN.

FIG. 15 depicts the results of an ELISA designed to detect darbepoetin alfa in culture media of primary human epidermal keratinocytes transfected with RNA comprising the indicated nucleotides and encoding NOVEPOETIN.

FIG. 16 depicts primary human dermal fibroblasts transfected with RNA comprising 5-methoxyuridine and encoding hTERT. Cells were fixed and stained using an antibody targeting hTERT 24 hours after transfection.

FIG. 17 depicts primary adult human dermal fibroblasts transfected with RNA encoding green fluorescent protein (“GFP”), prepared and stored as indicated.

FIG. 18 depicts single administration of NOVECRIT induced a rapid increase and sustained level of NOVEPOIETIN in serum. The Y axis shows concentration of NOVEPOIETIN protein (mU/mL).

FIG. 19 depicts a single administration of NOVECRIT stimulated erythropoiesis, yielding elevated hematocrit for at least 14 days. The left panel shows % hematocrit on the Y axis, while the right panel shows % reticulocytes.

FIG. 20 depicts a table summarizing TNFα, IL-6, and IFNα cytokine levels in plasma samples collected from a maximum tolerated dose of NOVECRIT in male Sprague Dawley rats study of Example 35.

FIG. 21 depicts a SURVEYOR assay using the DNA of primary adult human dermal fibroblasts transfected with RNA TALENs targeting the sequence TGAGCAGAAGTGGCTCAGTG (SEQ ID NO: 467) and TGGCTGTACAGCTACACCCC (SEQ ID NO: 468), located within the COL7A1 gene. The bands present in the +RNA lane indicate editing of a region of the gene that is frequently involved in dystrophic epidermolysis bullosa.

FIG. 22 depicts a SURVEYOR assay using the DNA of primary adult human dermal fibroblasts transfected with RNA TALENs targeting the sequence TTCCACTCCTGCAGGGCCCC (SEQ ID NO: 469) and TCGCCCTTCAGCCCGCGTTC (SEQ ID NO:470), located within the COL7A1 gene. The bands present in the +RNA lane indicate editing of a region of the gene that is frequently involved in dystrophic epidermolysis bullosa.

FIG. 23 shows the immunogenicity of various synthetic RNA constructs in the context of a gene-editing (i.e. unmodified nucleotides “A,G,U,C”; pseudouridine only “psU”; 5-methylcytidine only “5mC”; both pseudouridine and 5-methylcytidine “psU+5mC”; and a negative control “neg”).

FIG. 24 shows the gene-editing activity in cells transfected with various synthetic RNA constructs (i.e. unmodified nucleotides “A,G,U,C”; psuedouridine only “psU”; 5-methylcytidine only “5mC”; both psuedouridine and 5-methylcytidine “psU+5mC”; and a negative control “neg”).

FIG. 25 depicts gene editing of the COL7A1 gene in primary human epidermal keratinocytes transfected with RNA encoding TALENs and a single-stranded DNA repair template (“RT”) of the indicated length. The presence of bands at the locations shown by asterisks (“*”) indicates successful gene editing.

FIG. 26 depicts gene editing of the COL7A1 gene in primary human epidermal keratinocytes transfected with RNA encoding TALENs and an 80 nt single-stranded DNA repair template (“RT”) at the indicated ratios of RNA to repair template. The presence of bands at the locations shown by asterisks (“*”) indicates successful gene editing.

FIG. 27 depicts gene correction of the COL7A1 gene in primary human epidermal keratinocytes transfected with RNA encoding TALENs and a single-stranded DNA repair template (“RT”) of the indicated length. The presence of bands at the locations shown by asterisks (“*”) indicates successful gene correction.

FIG. 28 depicts gene correction of the COL7A1 gene in primary human epidermal keratinocytes transfected with RNA encoding TALENs and an 80 nt single-stranded DNA repair template (“RT”) at the indicated ratios of RNA to repair template. The presence of bands at the locations shown by asterisks (“*”) indicates successful gene correction.

FIG. 29 depicts gene editing (“T7E1”) and correction (“Digestion”) of the COL7A1 gene in primary human epidermal keratinocytes transfected with RNA encoding TALENs and an 80 nt single-stranded DNA repair template (“RT”). The presence of bands at the locations shown by asterisks (“*”) indicates successful gene editing (“T7E1”) and correction (“Digestion”).

FIG. 30 depicts the amount of BMP7 protein secreted by primary human dermal fibroblasts and primary human epidermal keratinocytes transfected with RNA encoding the indicated BMP7 variant, measured by ELISA. Asterisks (“*”) indicate saturation of the ELISA assay.

FIG. 31 depicts the amount of parathyroid hormone secreted by primary human epidermal keratinocytes transfected with RNA encoding PTH, measured by ELISA.

FIG. 32 demonstrates that repeated administration of NOVECRIT stimulated erythropoiesis, yielding elevated hematocrit for at least 14 days. For each data set, the order of histograms from left to right is: Group 1, Group 2, Group 3, Group 4, and Group 5.

FIG. 33 provides an illustrative experimental design for studying the effects of RNAs encoding BMP7 variants for the prevention and treatment of diabetic nephropathy.

FIG. 34 depicts BMP7 protein levels in rats treated with RNAs encoding BMP7 variants as described in Example 42. Error bars indicate SEM (n=6).

FIG. 35 depicts the urine volume, urine albumin, and urine creatinine levels in rats treated with RNAs encoding BMP7 variants as described in Example 42. For each data set, the order of histograms from left to right is: urine volume, urine creatinine, and urine albumin.

FIG. 36 depicts the effect of RNAs encoding BMP7 variants in treating diabetic nephropathy as described in Example 42. For each data set, the order of histograms from left to right is: Group 6—vehicle and Group 7—FTB-2 (BMP7 variant A).

FIG. 37, panels A-I, depict the results of gene-expression analysis of human epidermal keratinocytes transfected with RNA encoding BDNF, BMP-2, BMP-6, IL-2, IL-6, IL-15, IL-22, LIF or FGF-21.

FIG. 38 depicts the results of gene-expression analysis of human epidermal keratinocytes transfected with RNA encoding IL-15 or IL-15 and IL-15RA.

FIG. 39 depicts the serum levels of FGF21, IL15, IL6, IL22, and Novepoietin following a single intradermal injection of various RNAs encoding these proteins as described in Example 45. Three rats were analyzed for each RNA tested.

FIG. 40 depicts the results of the wound-healing assay described in Example 46. Four scratch locations were measured for each well, and two wells were measured for each condition. Error bars indicate standard deviation.

FIG. 41 depicts gene editing of primary human epidermal keratinocytes, as described in Example 47.

FIG. 42 depicts gene correction of primary human epidermal keratinocytes, as described in Example 47.

FIG. 43 depicts gene editing of primary human epidermal keratinocytes, as described in Example 47.

FIG. 44 depicts IL10 production in COLO 205 human colorectal adenocarcinoma cells. COLO 205 cells were plated at a density of 20,000 cells/well in a 24 well plate in 0.5 mL RPM11640+10% FBS per well. The next day, cells were either (i) transfected with 0.4 μg RNA encoding GFP, as described in example (Example 3), (ii) transfected with 0.4 μg RNA encoding IL22, as described in example (Example 3), (iii) exposed to cell culture medium taken from human epidermal keratinocytes transfected with RNA encoding IL22, (iv) exposed to cell culture medium taken from untransfected human epidermal keratinocytes, (v) exposed to recombinant human IL22 (782-IL-010; R&D Systems). 48 h later, IL10 levels in the culture medium were measured via ELISA (D1000B; R&D Systems). IL22 levels for sample (iii) were determined via ELISA (D2200; R&D Systems) prior to the experiment, and the same volumes of medium were used for condition (iv).

FIG. 45 depicts a BMP7 activity assay involving the measurement of alkaline phosphatase activity in ATDC 5 cells following transfection with RNA encoding BMP7 (variant A) or exposure to recombinant human BMP7. ATDC 5 cells were plated at a density of 20,000 cells/well in a 24 well plate in DMEM/F12+5% FBS. The next day, the cells were serum starved by changing the medium to DMEM/F12+2% FBS. The following day, the cells were either (i) transfected with RNA encoding BMP7 or (ii) exposed to recombinant human BMP7 (54-BP-010, R&D Systems). Three days later, intracellular alkaline phosphatase activity was measured (ab83369; Abcam).

FIG. 46 depicts the results of an experiment in which 100,000 primary human neonatal epidermal keratinocytes (animal-protein free) were transfected with 2 μg RNA encoding the indicated gene-editing proteins (1 μg L and 1 μg R). DNA was harvested after 48 h and analyzed for gene editing (A/B indicates RIBOSLICE_A L, RIBOSLICE_B R; RIBOSLICE A indicates repeat sequences comprising the sequence: GHGG, HG, GHGG, HG, etc.; RIBOSLICE B indicates repeat sequences comprising the sequence: HG, GHGG, HG, GHGG, etc.; L targets the sequence: TGCCTGGTCCCTGTCTCCCT (SEQ ID NO: 615); R targets the sequence: TGTCTTCTGGGCAGCATCTC (SEQ ID NO: 616); a target sequence is approximately 75 bp from A1AT [SERPINA1] start codon). “TAL” indicates a control TALEN directed to the target sequence.

FIG. 47 depicts the results of an experiment in which 100,000 primary human neonatal epidermal keratinocytes (animal-protein free) were transfected with 2 μg RNA encoding the indicated gene-editing proteins (1 μg L and 1 μg R). DNA was harvested after 48 h and analyzed for gene editing (A/B indicates RIBOSLICE_A L, RIBOSLICE_B R; RIBOSLICE A indicates repeat sequences comprising the sequence: GHGG, HG, GHGG, HG, etc.; RIBOSLICE B indicates repeat sequences comprising the sequence: HG, GHGG, HG, GHGG, etc.; L targets the sequence: TATTCCCGGGCTCCCAGGCA (SEQ ID NO: 622); R targets the sequence: TCTCCTGGCCTTCCTGCCTC (SEQ ID NO: 612); a target sequence is near the end of exon 73 of COL7A1). “TAL” indicates a control TALEN directed to the target sequence.

FIG. 48 depicts the results of an experiment in which 50,000 primary human neonatal epidermal keratinocytes (HEKn) (animal-protein free) were transfected with 2 μg RNA encoding the indicated gene-editing proteins. DNA was harvested after 48 h and analyzed for gene editing (“Neg” indicates untreated HEKn DNA; “WT” indicates wild type FokI; “EA” indicates enhanced activity FokI (S35P and K58E); “Het” indicates heterodimer (L: Q103E, N113D, I116L, R: E107K, H154R, 1155K); “EA/Het” indicates both EA and Het; L targets the sequence: TATTCCCGGGCTCCCAGGCA (SEQ ID NO: 622); R targets the sequence: TCTCCTGGCCTTCCTGCCTC (SEQ ID NO: 612); a target sequence is near the end of exon 73 of COL7A1).

FIG. 49 depicts the results of an experiment in which 100,000 primary human neonatal epidermal keratinocytes (animal-protein free) per well of a 6-well plate were transfected with 2 ug RNA encoding hGDF15. The culture medium was analyzed for GDF15 at the indicated time after transfection by ELISA (R&D DGD150).

FIG. 50 depicts the results of an experiment in which 100,000 primary human neonatal epidermal keratinocytes (animal-protein free) per well of a 6-well plate were transfected with 2 ug RNA encoding the indicated protein. The culture medium was analyzed for IFKB at the indicated time after transfection by ELISA (Abcam ab176644). For each time point, the histograms indicate (left to right): IFKB WT, IFKB S32A, S36A, and Neg.

FIG. 51 depicts the results of an experiment in which 100,000 primary human neonatal epidermal keratinocytes (animal-protein free) per well of a 6-well plate were transfected with 2 μg RNA encoding the indicated protein. Images were taken 24 h after transfection.

FIG. 52 depicts the results of an experiment in which 20,000 primary human neonatal epidermal keratinocytes (animal-protein free) per well of a 24-well plate were transfected with 0.2 μg RNA encoding SOD3. Cells were fixed and stained 24 h after transfection with a 1:100 dilution of NBP2-38493 (Novus) rabbit anti human SOD3 primary antibody and a 1:1000 dilution of 488 donkey anti-rabbit secondary antibody. “BF” indicates brightfield and “FL” indicates fluorescence.

FIG. 53 depicts the results of an experiment in which ZDF rats were administered RNA encoding hGDF15 by intradermal injection on Days 1, 8, and 15 (Group 4, grey curve (bottom)) or with vehicle only (Group 3, black curve (top)). ALT serum levels were measured at the indicated times.

FIG. 54 depicts the results of an experiment in which ZDF rats were administered RNA encoding hGDF15 by intradermal injection on Days 1, 8, and 15 (Group 4, grey curve (bottom)) or with vehicle only (Group 3, black curve (top)). AST serum levels were measured at the indicated times.

FIG. 55 depicts the results of an experiment in which ZDF rats were administered RNA encoding hGDF15 by intradermal injection on Days 1, 8, and 15 (Group 4, grey curve (top)) or with vehicle only (Group 3, black curve (bottom)). Total cholesterol serum levels were measured at the indicated times.

FIG. 56 depicts the results of an experiment in which ZDF rats were administered RNA encoding hGDF15 by intradermal injection on Days 1, 8, and 15 (Group 4, grey (bottom at day 7)) or with vehicle only (Group 3, black (top at day 7)). Glucose serum levels were measured at the indicated times.

FIG. 57 depicts the results of an experiment in which ZDF rats were administered RNA encoding hGDF15 by intradermal injection on Days 1, 8, and 15 (Group 4, grey (top)) or with vehicle only (Group 3, black (bottom)). Triglycerides serum levels were measured at the indicated times.

FIG. 58 depicts the results of an experiment in which ZDF rats were administered RNA encoding hGDF15 by intradermal injection on Days 1, 8, and 15 (treatment group) or with vehicle only (control group). % change of ALT, AST, Total cholesterol, Glucose, and Triglycerides serum levels in treatment group vs control group are shown.

FIG. 59 depicts the results of an experiment in which lean Sprague-Dawley rats were administered RNA encoding hGDF15 by intradermal injection on Days 1, 8, and 15 (Group 2) or with vehicle only (Group 1) and ZDF rats were administered RNA encoding hGDF15 by intradermal injection on Days 1, 8, and 15 (Group 4) or with vehicle only (Group 3). GDF15 serum levels were measured at the indicated times. For each timepoint, the histograms indicate (left to right) Group 1, Group 2, Group 3, and Group 4.

FIG. 60 depicts the results of an experiment in which 200,000 primary human neonatal epidermal keratinocytes (animal-protein free) per well of a 6-well plate were transfected with 2 μg RNA encoding hESM1. The culture medium was analyzed for ESM1 52 hours after transfection by ELISA (Abcam ab213776).

FIG. 61 depicts the results of an experiment in which 100,000 primary human neonatal epidermal keratinocytes (animal-protein free) were transfected with 2 μg RNA encoding the HBB exon 1 TALEN L and HBB exon 1 TALEN R gene-editing proteins (1 μg each). DNA was harvested after 48 h and analyzed for gene editing (T7E1 assay; forward primer: gccaaggacaggtacggctgtcatc (SEQ ID NO: 627); reverse primer: cttgccatgagccttcaccttagggttg (SEQ ID NO: 628); product size: 518 nt; predicted band sizes: 300 nt, 218 nt).

FIG. 62 depicts the results of an experiment in which 100,000 primary human neonatal epidermal keratinocytes (animal-protein free) were transfected with 2 μg RNA encoding the PD1 exon 1 TALEN L and PD1 exon 1 TALEN R gene-editing proteins (1 μg each). DNA was harvested after 48 h and analyzed for gene editing (T7E1 assay; forward primer: tcctctgtctccctgtctctgtctctctctc (SEQ ID NO: 594); reverse primer: ggacttgggccaggggaggag (SEQ ID NO: 595); product size: 612 nt; predicted band sizes: 349 nt, 263 nt).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the discovery of a safe and effective dosing strategy for nucleic acid drugs, including RNA, such as RNA comprising non-canonical (or “modified”) nucleotides, in humans. The inventors believe this to be the first report of safe and effective dosing of RNA molecules, including those comprising non-canonical nucleotides, in humans. Despite reports in the art that very large doses of RNA molecules are needed for mammalian dosing, and minimal therapeutic effect is achieved despite high dosing (see, e.g. US Patent Publication No. 2013/0245103), the present inventors have surprisingly managed to dose synthetic RNA in a human and achieve significant target protein expression with minimal immunological or other side effects.

In various embodiments, the present invention provides improved doses, formulations, administration and methods of use of nucleic acid drugs, which include RNA, which may contain non-canonical nucleotides (e.g. a residue other than adenine, guanine, thymine, uracil, and cytosine or the standard nucleoside, nucleotide, deoxynucleoside or deoxynucleotide derivatives thereof). In various embodiments, the RNA comprising non-canonical nucleotides leads to the expression of a protein encoded by the RNA, the protein often being one of therapeutic benefit (sometimes called the “target” or “protein of interest”). Further, this expression of therapeutic protein is achieved with minimal or negligible toxicity.

In various aspects, the present invention is based on the surprising discovery of safe and effective doses and administration parameters of nucleic acid drugs for human subjects. Nucleic acid drugs include a dsDNA molecule, a ssDNA molecule, a RNA molecule, a dsRNA molecule, a ssRNA molecule, a plasmid, an oligonucleotide, a synthetic RNA molecule, a miRNA molecule, an mRNA molecule, and an siRNA molecule. In various embodiments, the RNA comprises non-canonical nucleotides.

In some aspects, there is provided a method for delivering a nucleic acid drug, comprising administering an effective dose of a nucleic acid drug to a human subject in need thereof, wherein the nucleic acid drug comprises a synthetic RNA. In various embodiments, the effective dose is an amount sufficient to substantially increase an amount of a protein encoded by the nucleic acid drug in the human subject. For example, when the nucleic acid drug is a synthetic RNA comprising one or more modified nucleotides, the nucleic acid drug may result in higher protein expression than levels obtainable with a nucleic acid drug that does not comprise one or more modified nucleotides (e.g., RNA comprising the canonical nucleotides A, G, U, and C). In some embodiments, the nucleic acid drug results in about a 2-fold, or about a 3-fold, or about a 4-fold, or about a 5-fold, or about a 10-fold, or about a 15-fold, or about a 20-fold, or about a 25-fold, or about a 30-fold, or about a 35-fold, or about a 40-fold, or about a 45-fold, or about a 50-fold, or about a 100-fold increase in protein expression as compared to levels obtainable with a nucleic acid drug that does not comprise one or more modified nucleotides.

In some embodiments, the nucleic acid drug provides a sustained therapeutic effect that is optionally mediated by a sustained expression of target protein. For instance, in some embodiments, the therapeutic effect is present for over about 1 day, or over about 2 days, or over about 3 days, or over about 4 days, or over about 5 days, or over about 6 days, or over about 7 days, or over about 8 days, or over about 9 days, or over about 10 days, or over about 14 days after administration. In some embodiments, this sustained effect obviates the need for, or reduces the amount of, maintenance doses.

In some embodiments, the nucleic acid drug provides a sustained target protein level. For instance, in some embodiments, the target protein is present (e.g. in measurable amounts, e.g. in the serum of a patient to whom the nucleic acid drug has been administered) for over about 1 day, or over about 2 days, or over about 3 days, or over about 4 days, or over about 5 days, or over about 6 days, or over about 7 days, or over about 8 days, or over about 9 days, or over about 10 days, or over about 14 days after administration. In some embodiments, this sustained effect obviates the need for, or reduces the amount of, maintenance doses.

In various embodiments, the nucleic acid drug provides therapeutic action without sustained presence of the nucleic acid drug itself. In some embodiments, the nucleic acid drug is rapidly metabolized, for instance, within about 6 hours, or about 12 hours, or about 18 hours, or about 24 hours, or about 2 days, or about 3 days, or about 4 days, or about 5 days, or about 1 week from administration.

In various embodiments, the effective dose is an amount that substantially avoids cell toxicity in vivo. In various embodiments, the effective dose is an amount that substantially avoids an immune reaction in a human subject. For example, the immune reaction may be an immune response mediated by the innate immune system. Immune response can be monitored using markers known in the art (e.g. cytokines, interferons, TLRs). In some embodiments, the effective dose obviates the need for treatment of the human subject with immune suppressants agents (e.g. B18R) used to moderate the residual toxicity. Accordingly, in some embodiments, the present methods allow for dosing that provides increased protein expression and reduces toxicity.

In some embodiments, the immune response is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 99.9%, or greater than about 99.9% as compared to the immune response induced by a corresponding unmodified nucleic acid. In some embodiments, upregulation of one or more immune response markers is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, about 99.9%, or greater than about 99.9% as compared to the upregulation of the one or more immune response markers induced by a corresponding unmodified nucleic acid. In some embodiments, the immune response marker comprises an mRNA or protein product of an interferon gene, including an interferon alpha gene, IFNB1, TLR3, RARRES3, EIF2AK2, STAT1, STAT2, IFIT1, IFIT2, IFIT3, IFIT5, OAS1, OAS2, OAS3, OASL, ISG20 or a fragment, variant, analogue, or family-member thereof. In some embodiments, the immune response marker comprises an mRNA or protein product of an TNF gene, including an TNF alpha gene, TNFRSF1A; TNFRSF1B; LTBR; TNFRSF4; CD40; FAS; TNFRSF6B; CD27; TNFRSF8; TNFRSF9; TNFRSF10A; TNFRSF10B; TNFRSF10C; TNFRSF10D; TNFRSF11A; TNFRSF11B; TNFRSF12A; TNFRSF13B; TNFRSF13C; TNFRSF14; NGFR; TNFRSF17; TNFRSF18; TNFRSF19; TNFRSF21; TNFRSF25; and EDA2R or a fragment, variant, analogue, or family-member thereof. In some embodiments, the immune response marker comprises an mRNA or protein product of an interleukin gene, including an IL-6 gene, IL-1; IL-2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8 or CXCL8; IL-9; IL-10; IL-11; IL-12; IL-13; IL-14; IL-15; IL-16; IL-17; IL-18; IL-19; IL-20; IL-21; IL-22; IL-23; IL-24; IL-25; IL-26; IL-27; IL-28; IL-29; IL-30; IL-31; IL-32; IL-33; IL-35; IL-36 or a fragment, variant, analogue, or family-member thereof.

In some embodiments, cell death is about 10%, about 25%, about 50%, about 75%, about 85%, about 90%, about 95%, or over about 95% less than the cell death observed with a corresponding unmodified nucleic acid. Moreover, cell death may affect fewer than about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 1%, about 0.1%, about 0.01% or fewer than about 0.01% of cells contacted with the modified nucleic acids.

In some embodiments, there is provided a method for expressing a protein of interest in a population of cells in a mammalian subject, comprising administering a non-viral transfection composition comprising an effective dose of a RNA encoding the protein of interest to said cells, the RNA containing one or more non-canonical nucleotides that avoid substantial cellular toxicity, where the transfection composition is administered in an amount that allows for expression of said protein in said cells for at least about five days (e.g. about 5, or about 6, or about 7, about 8, or about 9, or about 10, or about 14 days) without substantial cellular toxicity. In some embodiments, there is provided a method for expressing a protein of interest in a population of cells in a mammalian subject, comprising administering a non-viral transfection composition comprising an effective dose of a RNA encoding the protein of interest to said cells, the RNA containing one or more non-canonical nucleotides that avoid substantial cellular toxicity, where the transfection composition is administered in an amount that allows for expression of said protein in said cells for at least about six hours (e.g. about six hours, or about 12 hours, or about 1 day, or about 2 days, or about 3 days, or about 4 days, or about 5 days) without substantial cellular toxicity.

In some embodiments, the effective dose of the nucleic acid drug, including synthetic RNA, is about 100 ng to about 2000 ng, or about 200 ng to about 1900 ng, or about 300 ng to about 1800 ng, or about 400 ng to about 1700 ng, or about 500 ng to about 1600 ng, or about 600 ng to about 1500 ng, or about 700 ng to about 1400 ng, or about 800 ng to about 1300 ng, or about 900 ng to about 1200 ng, or about 1000 ng to about 1100 ng, or about 500 ng to about 2000 ng, or about 500 ng to about 1500 ng, or about 500 ng to about 1000 ng, or about 1000 ng to about 1500 ng, or about 1000 ng to about 2000 ng, or about 1500 ng to about 2000 ng, or about 100 ng to about 500 ng, or about 200 ng to about 400 ng, or about 10 ng to about 100 ng, or about 20 ng to about 90 ng, or about 30 ng to about 80 ng, or about 40 ng to about 70 ng, or about 50 ng to about 60 ng.

In some embodiments, the effective dose of the nucleic acid drug, including synthetic RNA, is no more than about 50 ng, or about 100 ng, or about 200 ng, or about 300 ng, or about 400 ng, or about 500 ng, or about 600 ng, or about 700 ng, or about 800 ng, or about 900 ng, or about 1000 ng, or about 1100 ng, or about 1200 ng, or about 1300 ng, or about 1400 ng, or about 1500 ng, or about 1600 ng, or about 1700 ng, or about 1800 ng, or about 1900 ng, or about 2000 ng, or about 3000 ng, or about 4000 ng, or about 5000 ng.

In some embodiments, the effective dose of the nucleic acid drug, including synthetic RNA, is about 50 ng, or about 100 ng, or about 200 ng, or about 300 ng, or about 400 ng, or about 500 ng, or about 600 ng, or about 700 ng, or about 800 ng, or about 900 ng, or about 1000 ng, or about 1100 ng, or about 1200 ng, or about 1300 ng, or about 1400 ng, or about 1500 ng, or about 1600 ng, or about 1700 ng, or about 1800 ng, or about 1900 ng, or about 2000 ng, or about 3000 ng, or about 4000 ng, or about 5000 ng.

In some embodiments, the effective dose of the nucleic acid drug, including synthetic RNA, is about 0.028 pmol, or about 0.05 pmol, or about 0.1 pmol, or about 0.2 pmol, or about 0.3 pmol, or about 0.4 pmol, or about 0.5 pmol, or about 0.6 pmol, or about 0.7 pmol, or about 0.8 pmol, or about 0.9 pmol, or about 1.0 pmol, or about 1.2 pmol, or about 1.4 pmol, or about 1.6 pmol, or about 1.8 pmol, or about 2.0 pmol, or about 2.2 pmol, or about 2.4 pmol, or about 2.6 pmol, or about 2.8 pmol, or about 3.0 pmol, or about 3.2 pmol, or about 3.4 pmol, or about 3.6 pmol, or about 3.8 pmol, or about 4.0 pmol, or about 4.2 pmol, or about 4.4 pmol, or about 4.6 pmol, or about 4.8 pmol, or about 5.0 pmol, or about 5.5 pmol, or about 5.7 pmol.

In some embodiments, the nucleic acid drug, including synthetic RNA, is administered at a concentration of about 0.1 nM, or about 0.25 nM, or about 0.5 nM, or about 0.75 nM, or about 1 nM, or about 2.5 nM, or about 5 nM, or about 7.5 nM, or about 10 nM, or about 20 nM, or about 30 nM, or about 40 nM, or about 50 nM, or about 60 nM, or about 70 nM, or about 80 nM, or about 90 nM, or about 100 nM, or about 110 nM, or about 120 nM, or about 150 nM, or about 175 nM, or about 200 nM.

In some embodiments, the effective dose of the nucleic acid drug is about 350 ng/cm², or about 500 ng/cm², or about 750 ng/cm², or about 1000 ng/cm², or about 2000 ng/cm², or about 3000 ng/cm², or about 4000 ng/cm², or about 5000 ng/cm², or about 6000 ng/cm², or about 7000 ng/cm². In other embodiments, the effective dose is less than about 350 ng/cm². In certain embodiments, the effective dose is about 35 ng/cm², or about 50 ng/cm², or about 75 ng/cm², or about 100 ng/cm², or about 150 ng/cm², or about 200 ng/cm², or about 250 ng/cm², or about 300 ng/cm², or about 350 ng/cm².

In some embodiments, the effective dose of the nucleic acid drug is about 35 ng/cm² to about 7000 ng/cm², or about 50 ng/cm² to about 5000 ng/cm², or about 100 ng/cm² to about 3000 ng/cm², or about 500 ng/cm² to about 2000 ng/cm², or about 750 ng/cm² to about 1500 ng/cm², or about 800 ng/cm² to about 1200 ng/cm², or about 900 ng/cm² to about 1100 ng/cm².

In some embodiments, the effective dose of the nucleic acid drug is about 1 picomole/cm², or about 2 picomoles/cm², or about 3 picomoles/cm², or about 4 picomoles/cm², or about 5 picomoles/cm², or about 6 picomoles/cm², or about 7 picomoles/cm², or about 8 picomoles/cm², or about 9 picomoles/cm², or about 10 picomoles/cm², or about 12 picomoles/cm², or about 14 picomoles/cm², or about 16 picomoles/cm², or about 18 picomoles/cm², or about 20 picomoles/cm². In other embodiments, the effective dose is less than about 1 picomole/cm². In certain embodiments, the effective dose is about 0.1 picomoles/cm², or about 0.2 picomoles/cm², or about 0.3 picomoles/cm², or about 0.4 picomoles/cm², or about 0.5 picomoles/cm², or about 0.6 picomoles/cm², or about 0.7 picomoles/cm², or about 0.8 picomoles/cm², or about 0.9 picomoles/cm², or about 1 picomole/cm².

In some embodiments, the effective dose of the nucleic acid drug is about 0.1 picomoles/cm² to about 20 picomoles/cm², or about 0.2 picomoles/cm² to about 15 picomoles/cm², or about 0.5 picomoles/cm² to about 10 picomoles/cm², or about 0.8 picomoles/cm² to about 8 picomoles/cm², or about 1 picomole/cm² to about 5 picomoles/cm², or about 2 picomoles/cm² to about 4 picomoles/cm².

In various embodiments, the nucleic acid drug, including synthetic RNA, is administered in a pharmaceutically acceptable formulation. In various embodiments, the nucleic acid drug, including synthetic RNA, is formulated for one or more of injection and topical administration. By way of example, the nucleic acid drug, including synthetic RNA, may be formulated for injection to a tissue of interest, e.g. a disease site (by way of non-limiting example, a tumor). In various embodiments, injection includes delivery via a patch. In some embodiments, the delivery is mediated by electrical stimulation. In various embodiments, the nucleic acid drug, including synthetic RNA, is formulated for administration to one or more of the epidermis (optionally selected from the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum germinativum), basement membrane, dermis (optionally selected from the papillary region and the reticular region), subcutis, conjunctiva cornea, sclera, iris, lens, corneal limbus, optic nerve, choroid, ciliary body, anterior segment, anterior chamber, and retina. In various embodiments, the nucleic acid drug, including synthetic RNA, is formulated for one or more of subcutaneous injection, intradermal injection, subdermal injection, intramuscular injection, intraocular injection, intravitreal injection, intra-articular injection, intracardiac injection, intravenous injection, epidural injection, intrathecal injection, intraportal injection, intratumoral injection, and topical administration. In various embodiments, the nucleic acid drug, including synthetic RNA, is formulated for intradermal (ID) injection to one or more of the dermis or epidermis. In various embodiments, the nucleic acid drug, including synthetic RNA, is administered in a manner such that it effects one or more of keratinocytes and fibroblasts (e.g. causes these cells to express one or more therapeutic proteins).

Accordingly, the present invention provides various formulations as described herein. Further, in some embodiments, the formulations described herein find use in the various delivery and/or treatment methods of the present invention. For instance, formulations can comprise a vesicle, for instance, a liposome (see Langer, 1990, Science 249:1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989). In various embodiments, the formulation comprises an aqueous suspension of liposomes. Illustrative liposome components are set forth in Table 1, and are given by way of example, and not by way of limitation. In various embodiments, one or more, or two or more, or three or more, or four or more, or five or more of the lipids of Table 1 are combined in a formulation.

TABLE 1 Illustrative Biocompatible Lipids 1 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-Cholesterol) 2 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP/18:1 TAP) 3 N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan- 1-aminium (DOBAQ) 4 1,2-dimyristoyl-3-trimethylammonium-propane (14:0 TAP) 5 1,2-dipalmitoyl-3-trimethylammonium-propane (16:0 TAP) 6 1,2-stearoyl-3-trimethylammonium-propane (18:0 TAP) 7 1,2-dioleoyl-3-dimethylammonium-propane (DODAP/18:1 DAP) 8 1,2-dimyristoyl-3-dimethylammonium-propane (14:0 DAP) 9 1,2-dipalmitoyl-3-dimethylammonium-propane (16:0 DAP) 10 1,2-distearoyl-3-dimethylammonium-propane (18:0 DAP) 11 dimethyldioctadecylammonium (18:0 DDAB) 12 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (12:0 EthylPC) 13 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:0 EthylPC) 14 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1 EthylPC) 15 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (16:0 EthylPC) 16 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (18:0 EthylPC) 17 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1 EthylPC) 18 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:1-18:1 EthylPC) 19 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) 20 N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino- propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5) 21 2,3-dioleyloxy-N-[2-spermine carboxamide]ethyl-N,N-dimethyl-1- propanammonium trifluoroacetate (DOSPA) 22 1,3-di-oleoyloxy-2-(6-carboxy-spermyl)-propylamid (DOSPER) 23 N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2- hydroxyethyl)ammonium bromide (DMRIE) 24 LIPOFECTAMINE, LIPOFECTAMINE 2000, LIPOFECTAMINE RNAiMAX, LIPOFECTAMINE 3000, LIPOFECTAMINE MessengerMAX, TransIT mRNA 25 dioctadecyl amidoglyceryl spermine (DOGS) 26 dioleoyl phosphatidyl ethanolamine (DOPE)

In some embodiments, the liposomes include LIPOFECTAMINE 3000. In some embodiments, the liposomes include one or more lipids described in U.S. Pat. No. 4,897,355 or 7,479,573 or in International Patent Publication No. WO/2015/089487, or in Feigner, P. L. et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7417, the entire contents of each is incorporated by reference in their entireties).

In some embodiments, the liposome comprises N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). In some embodiments, the liposome comprises dioleoylphosphatidylethanolamine (DOPE).

In one embodiment, the liposomes include one or more polyethylene glycol (PEG) chains, optionally selected from PEG200, PEG300, PEG400, PEG600, PEG800, PEG1000, PEG1500, PEG2000, PEG3000, and PEG4000. In some embodiments, the PEG is PEG2000. In some embodiments, the liposomes include 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) or a derivative thereof.

In some embodiments, the formulation comprises one or more of N-(carbonyl-ethoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (MPEG2000-DSPE), fully hydrogenated phosphatidylcholine, cholesterol, LIPOFECTAMINE 3000, a cationic lipid, a polycationic lipid, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-5000] (FA-MPEG5000-DSPE).

In one embodiment, the formulation comprises about 3.2 mg/mL N-(carbonyl-ethoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (MPEG2000-DSPE), about 9.6 mg/mL fully hydrogenated phosphatidylcholine, about 3.2 mg/mL cholesterol, about 2 mg/mL ammonium sulfate, and histidine as a buffer, with about 0.27 mg/mL 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-5000](FA-MPEG5000-DSPE) added to the lipid mixture. In another embodiment, the nucleic acids are complexed by combining 1 μL of LIPOFECTAMINE 3000 per about 1 μg of nucleic acid and incubating at room temperature for at least about 5 minutes. In one embodiment, the LIPOFECTAMINE 3000 is a solution comprising a lipid at a concentration of about 1 mg/mL. In some embodiments, nucleic acids are encapsulated by combining about 10 μg of the liposome formulation per about 1 μg of nucleic acid and incubating at room temperature for about 5 minutes.

In some embodiments, the formulation comprises one or more nanoparticles. In one embodiment, the nanoparticle is a polymeric nanoparticle. In various embodiments, the formulation comprises one or more of a diblock copolymer, a triblock copolymer, a tetrablock copolymer, and a multiblock copolymer. In various embodiments, the formulation comprises one or more of polymeric nanoparticles comprising a polyethylene glycol (PEG)-modified polylactic acid (PLA) diblock copolymer (PLA-PEG) and PEG-polypropylene glycol-PEG-modified PLA-tetrablock copolymer (PLA-PEG-PPG-PEG).

In some embodiments, the formulation comprises one or more lipids that are described in WO/2000/027795, the entire contents of which are hereby incorporated by reference.

In one embodiment, the therapeutic comprises one or more ligands. In another embodiment, the therapeutic comprises at least one of: androgen, CD30 (TNFRSF8), a cell-penetrating peptide, CXCR, estrogen, epidermal growth factor, EGFR, HER2, folate, insulin, insulin-like growth factor-I, interleukin-13, integrin, progesterone, stromal-derived-factor-1, thrombin, vitamin D, and transferrin or a biologically active fragment or variant thereof.

The active compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention may be via any common route so long as the target tissue is available via that route. This includes oral, nasal, or buccal. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal, intraportall or intravenous injection, or by direct injection into diseased, e.g. cancer, tissue. The agents disclosed herein may also be administered by catheter systems. Such compositions would normally be administered as pharmaceutically acceptable compositions as described herein.

Administration of the compositions described herein may be, for example, by injection, topical administration, ophthalmic administration and intranasal administration. The injection, in some embodiments, may be linked to an electrical force (e.g. electroporation, including with devices that find use in electrochemotherapy (e.g. CLINIPORATOR, IGEA SrI, Carpi [MO], Italy)). The topical administration may be, but is not limited to, a cream, lotion, ointment, gel, spray, solution and the like. The topical administration may further include a penetration enhancer such as, but not limited to, surfactants, fatty acids, bile salts, chelating agents, non-chelating non-surfactants, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether, fatty acids and/or salts in combination with bile acids and/or salts, sodium salt in combination with lauric acid, capric acid and UDCA, and the like. The topical administration may also include a fragrance, a colorant, a sunscreen, an antibacterial and/or a moisturizer. The compositions described herein may be administered to at least one site such as, but not limited to, forehead, scalp, hair follicles, hair, upper eyelids, lower eyelids, eyebrows, eyelashes, infraorbital area, periorbital areas, temple, nose, nose bridge, cheeks, tongue, nasolabial folds, lips, periobicular areas, jaw line, ears, neck, breast, forearm, upper arm, palm, hand, finger, nails, back, abdomen, sides, buttocks, thigh, calf, feet, toes and the like.

Routes of administration include, for example: intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intracerebral, intravaginal, transdermal, intraportal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin. In some embodiments, the administering is effected orally or by parenteral injection.

Upon formulation, solutions may be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective, as described herein. The formulations may easily be administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution generally is suitably buffered and the liquid diluent first rendered isotonic with, for example, sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Preferably, sterile aqueous media are employed as is known to those of skill in the art, particularly in light of the present disclosure.

In various embodiments, the nucleic acid drug, including RNA comprising one or more non-canonical nucleotides, and/or formulations comprising the same, is administered locally, optionally by one or more of subcutaneous injection, intradermal injection, subdermal injection and intramuscular injection, and the effective dose is administered to a surface area of about 4 mm² to about 150 mm² (e.g. about, or no more than about, 4 mm², or about 5 mm², or about 6 mm², or about 7 mm², or about 8 mm², or about 10 mm², or about 20 mm², or about 50 mm², or about 100 mm², or about 150 mm²). In various embodiments, the nucleic acid drug, including RNA comprising one or more non-canonical nucleotides, and/or formulations comprising the same, is administered locally, optionally by one or more of subcutaneous injection, intradermal injection, subdermal injection and intramuscular injection, and the effective dose administered to a surface area of no more than about 4 mm², or about 5 mm², or about 6 mm², or about 7 mm², or about 8 mm², or about 10 mm², or about 20 mm², or about 50 mm², or about 100 mm², or about 150 mm². In various embodiments, the nucleic acid drug, including RNA comprising one or more non-canonical nucleotides, and/or formulations comprising the same, is administered locally, optionally by one or more of subcutaneous injection, intradermal injection, subdermal injection and intramuscular injection, and the effective dose administered to a surface area of about 4 mm², or about 5 mm², or about 6 mm², or about 7 mm², or about 8 mm², or about 10 mm², or about 20 mm², or about 50 mm², or about 100 mm², or about 150 mm².

In various embodiments, the nucleic acid drug, including RNA comprising one or more non-canonical nucleotides, and/or formulations comprising the same, is administered locally, optionally by one or more of subcutaneous injection, intradermal injection, subdermal injection and intramuscular injection, and the effective dose (weight RNA/surface area of injection) is about 35 ng/cm² to about 7000 ng/cm². In various embodiments, the nucleic acid drug, including RNA comprising one or more non-canonical nucleotides, and/or formulations comprising the same, is administered locally, optionally by one or more of subcutaneous injection, intradermal injection, subdermal injection and intramuscular injection, and the effective dose (weight RNA/surface area of injection) is no more than about 35 ng/cm², or about 50 ng/cm², or about 75 ng/cm², or about 100 ng/cm², or about 125 ng/cm², or about 150 ng/cm², or about 175 ng/cm², or about 200 ng/cm², or about 225 ng/cm², or about 250 ng/cm², or about 500 ng/cm², or about 1000 ng/cm², or about 2000 ng/cm², or about 5000 ng/cm², or about 7000 ng/cm². In various embodiments, the nucleic acid drug, including RNA comprising one or more non-canonical nucleotides, and/or formulations comprising the same, is administered locally, optionally by one or more of subcutaneous injection, intradermal injection, subdermal injection and intramuscular injection, and the effective dose (weight RNA/surface area of injection) is about 35 ng/cm², or about 50 ng/cm², or about 75 ng/cm², or about 100 ng/cm², or about 125 ng/cm², or about 150 ng/cm², or about 175 ng/cm², or about 200 ng/cm², or about 225 ng/cm², or about 250 ng/cm², or about 500 ng/cm², or about 1000 ng/cm², or about 2000 ng/cm², or about 5000 ng/cm², or about 7000 ng/cm².

Pharmaceutical preparations may additionally comprise delivery reagents (a.k.a. “transfection reagents”, a.k.a. “vehicles”, a.k.a. “delivery vehicles”) and/or excipients. Pharmaceutically acceptable delivery reagents, excipients, and methods of preparation and use thereof, including methods for preparing and administering pharmaceutical preparations to patients (a.k.a. “subjects”) are well known in the art, and are set forth in numerous publications, including, for example, in US Patent Appl. Pub. No. US 2008/0213377, the entirety of which is incorporated herein by reference.

For example, the present compositions can be in the form of pharmaceutically acceptable salts. Such salts include those listed in, for example, J. Pharma. Sci. 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use. P. H. Stahl and C. G. Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, which are hereby incorporated by reference in their entirety. Non-limiting examples of pharmaceutically acceptable salts include: sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, isobutyrate, phenylbutyrate, α-hydroxybutyrate, butyne-1,4-dicarboxylate, hexyne-1,4-dicarboxylate, caprate, caprylate, cinnamate, glycollate, heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, phthalate, teraphthalate, propiolate, propionate, phenylpropionate, sebacate, suberate, p-bromobenzenesulfonate, chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate, methylsulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, naphthalene-1,5-sulfonate, xylenesulfonate, tartarate salts, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metal such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, and organic amines, such as unsubstituted or hydroxy-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-lower alkylamines), such as mono-; bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxyl-lower alkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine, lysine, and the like.

The present pharmaceutical compositions can comprises excipients, including liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical excipients can be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like. In addition, auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used. In one embodiment, the pharmaceutically acceptable excipients are sterile when administered to a subject. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Any agent described herein, if desired, can also comprise minor amounts of wetting or emulsifying agents, or pH buffering agents.

Dosage forms suitable for parenteral administration (e.g. subcutaneous, intradermal, subdermal, intramuscular, intravenous, intraperitoneal, intra-articular, and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions (e.g. lyophilized composition), which can be dissolved or suspended in sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art.

In some embodiments, the formulations described herein may comprise albumin and a nucleic acid molecule.

In some embodiments, the invention relates to a cosmetic composition. In one embodiment, the cosmetic composition comprises albumin. In another embodiment, the albumin is treated with an ion-exchange resin or charcoal. In yet another embodiment, the cosmetic composition comprises a nucleic acid molecule. In a further embodiment, the cosmetic composition comprises both albumin and a nucleic acid molecule. Still other embodiments are directed to a cosmetic treatment article comprising a cosmetic composition contained in a device configured to deliver the composition to a patient. Still other embodiments are directed to a device configured to deliver a cosmetic composition to a patient. In one embodiment, the nucleic acid molecule encodes a member of the group: elastin, collagen, tyrosinase, melanocortin 1 receptor, keratin, filaggren, an antibody, and hyaluronan synthase or a biologically active fragment, variant, analogue or family member thereof.

In some embodiments, the present invention provides treatment regimens. The inventors have discovered that the doses and administration described herein can produce a substantial protein expression effect quickly (e.g. in about 6, or about 12, or about 24, or about 36, or about 48 hours). Further, these effects can be sustained for about 7 days, or longer. In some embodiments, the present methods provide for administration of a nucleic acid drug, including RNA comprising one or more non-canonical nucleotides, about weekly to about once every 20 weeks.

In some embodiments, the nucleic acid drug, including RNA comprising one or more non-canonical nucleotides, is administered about weekly, for at least 2 weeks (e.g. 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 weeks). In some embodiments, the nucleic acid drug, including RNA comprising one or more non-canonical nucleotides, is administered about every other week for at least one month (e.g. 1, or 2, or 3, or 4, or 5, or 6, or 12 months). In some embodiments, the nucleic acid drug, including RNA comprising one or more non-canonical nucleotides, is administered monthly or about every other month. In some embodiments, the nucleic acid drug, including RNA comprising one or more non-canonical nucleotides, is administered is administered for at least two months, or at least 4 months, or at least 6 months, or at least 9 months, or at least one year.

In some embodiments, the nucleic acid drug, including RNA comprising one or more non-canonical nucleotides, is administered about weekly, or about once every 2 weeks, or about once every 3 weeks, or about once every 4 weeks, or about once every 5 weeks, or about once every 6 weeks, or about once every 7 weeks, or about once every 8 weeks, or about once every 9 weeks, or about once every 10 weeks, or about once every 11 weeks, or about once every 12 weeks, or about once every 13 weeks, or about once every 14 weeks, or about once every 15 weeks, or about once every 20 weeks, or about once every 24 weeks.

In some embodiments, the nucleic acid drug, including RNA comprising one or more non-canonical nucleotides, is administered no more than about weekly, or about once every 2 weeks, or about once every 3 weeks, or about once every 4 weeks, or about once every 5 weeks, or about once every 6 weeks, or about once every 7 weeks, or about once every 8 weeks, or about once every 9 weeks, or about once every 10 weeks, or about once every 11 weeks, or about once every 12 weeks, or about once every 13 weeks, or about once every 14 weeks, or about once every 15 weeks, or about once every 20 weeks, or about 24 weeks.

Certain proteins have long half-lives, and can persist in tissues for several hours, days, weeks, months or years. It has now been discovered that certain methods of treating a patient can result in accumulation of one or more proteins, including, for example, one or more beneficial proteins. Certain embodiments are therefore directed to a method for treating a patient comprising delivering to a patient in a series of doses a nucleic acid encoding one or more proteins. In one embodiment the nucleic acid comprises RNA comprising one or more non-canonical nucleotides. In another embodiment, a first dose is given at a first time-point. In yet another embodiment, a second dose is given at a second time-point. In a further embodiment, the amount of at least one of the one or more proteins in the patient at the second time-point is greater than the amount of said protein at the first time-point. In a still further embodiment, the method results in the accumulation of said protein in the patient.

In various embodiments, the present invention relates to nucleic acid drugs, which, in various embodiments are RNA comprising one or more non-canonical nucleotides. Certain non-canonical nucleotides, when incorporated into RNA molecules, can reduce the toxicity of the RNA molecules, in part, without wishing to be bound by theory, by interfering with binding of proteins that detect exogenous nucleic acids, for example, protein kinase R, Rig-1 and the oligoadenylate synthetase family of proteins. Non-canonical nucleotides that have been reported to reduce the toxicity of RNA molecules when incorporated therein include: pseudouridine, 5-methyluridine, 2-thiouridine, 5-methylcytidine, N6-methyladenosine, and certain combinations thereof. However, the chemical characteristics of non-canonical nucleotides that can enable them to lower the in vivo toxicity of RNA molecules have, until this point, remained unknown. Furthermore, incorporation of large amounts of most non-canonical nucleotides, for example, 5-methyluridine, 2-thiouridine, 5-methylcytidine, and N6-methyladenosine, can reduce the efficiency with which RNA molecules can be translated into protein, limiting the utility of RNA molecules containing these nucleotides in applications that require protein expression. In addition, while pseudouridine can be completely substituted for uridine in RNA molecules without reducing the efficiency with which the synthetic RNA molecules can be translated into protein, in certain situations, for example, when performing frequent, repeated transfections, synthetic RNA molecules containing only adenosine, guanosine, cytidine, and pseudouridine can exhibit excessive toxicity.

It has now been discovered that, and in some embodiments the invention pertains to, RNA molecules containing one or more non-canonical nucleotides that include one or more substitutions at the 2C and/or 4C and/or 5C positions in the case of a pyrimidine or the 6C and/or 7N and/or 8C positions in the case of a purine can be less toxic than synthetic RNA molecules containing only canonical nucleotides, due in part to the ability of substitutions at these positions to interfere with recognition of synthetic RNA molecules by proteins that detect exogenous nucleic acids, and furthermore, that substitutions at these positions can have minimal impact on the efficiency with which the synthetic RNA molecules can be translated into protein, due in part to the lack of interference of substitutions at these positions with base-pairing and base-stacking interactions.

Examples of non-canonical nucleotides that include one or more substitutions at the 2C and/or 4C and/or 5C positions in the case of a pyrimidine or the 6C and/or 7N and/or 8C positions in the case of a purine include, but are not limited to: 2-thiouridine, 5-azauridine, pseudouridine, 4-thiouridine, 5-methyluridine, 5-methylpseudouridine, 5-aminouridine, 5-aminopseudouridine, 5-hydroxyuridine, 5-hydroxypseudouridine, 5-methoxyuridine, 5-methoxypseudouridine, 5-hydroxymethyluridine, 5-hydroxymethylpseudouridine, 5-carboxyuridine, 5-carboxypseudouridine, 5-formyluridine, 5-formylpseudouridine, 5-methyl-5-azauridine, 5-amino-5-azauridine, 5-hydroxy-5-azauridine, 5-methylpseudouridine, 5-aminopseudouridine, 5-hydroxypseudouridine, 4-thio-5-azauridine, 4-thiopseudouridine, 4-thio-5-methyluridine, 4-thio-5-aminouridine, 4-thio-5-hydroxyuridine, 4-thio-5-methyl-5-azauridine, 4-thio-5-amino-5-azauridine, 4-thio-5-hydroxy-5-azauridine, 4-thio-5-methylpseudouridine, 4-thio-5-aminopseudouridine, 4-thio-5-hydroxypseudouridine, 2-thiocytidine, 5-azacytidine, pseudoisocytidine, N4-methylcytidine, N4-aminocytidine, N4-hydroxycytidine, 5-methylcytidine, 5-aminocytidine, 5-hydroxycytidine, 5-methoxycytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytydine, 5-methyl-5-azacytidine, 5-amino-5-azacytidine, 5-hydroxy-5-azacytidine, 5-methylpseudoisocytidine, 5-aminopseudoisocytidine, 5-hydroxypseudoisocytidine, N4-methyl-5-azacytidine, N4-methylpseudoisocytidine, 2-thio-5-azacytidine, 2-thiopseudoisocytidine, 2-thio-N4-methylcytidine, 2-thio-N4-aminocytidine, 2-thio-N4-hydroxycytidine, 2-thio-5-methylcytidine, 2-thio-5-aminocytidine, 2-thio-5-hydroxycytidine, 2-thio-5-methyl-5-azacytidine, 2-thio-5-amino-5-azacytidine, 2-thio-5-hydroxy-5-azacytidine, 2-thio-5-methylpseudoisocytidine, 2-thio-5-aminopseudoisocytidine, 2-thio-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-azacytidine, 2-thio-N4-methylpseudoisocytidine, N4-methyl-5-methylcytidine, N4-methyl-5-aminocytidine, N4-methyl-5-hydroxycytidine, N4-methyl-5-methyl-5-azacytidine, N4-methyl-5-amino-5-azacytidine, N4-methyl-5-hydroxy-5-azacytidine, N4-methyl-5-methylpseudoisocytidine, N4-methyl-5-aminopseudoisocytidine, N4-methyl-5-hydroxypseudoisocytidine, N4-amino-5-azacytidine, N4-aminopseudoisocytidine, N4-amino-5-methylcytidine, N4-amino-5-aminocytidine, N4-amino-5-hydroxycytidine, N4-amino-5-methyl-5-azacytidine, N4-amino-5-amino-5-azacytidine, N4-amino-5-hydroxy-5-azacytidine, N4-amino-5-methylpseudoisocytidine, N4-amino-5-aminopseudoisocytidine, N4-amino-5-hydroxypseudoisocytidine, N4-hydroxy-5-azacytidine, N4-hydroxypseudoisocytidine, N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, N4-hydroxy-5-hydroxycytidine, N4-hydroxy-5-methyl-5-azacytidine, N4-hydroxy-5-amino-5-azacytidine, N4-hydroxy-5-hydroxy-5-azacytidine, N4-hydroxy-5-methylpseudoisocytidine, N4-hydroxy-5-aminopseudoisocytidine, N4-hydroxy-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-methylcytidine, 2-thio-N4-methyl-5-aminocytidine, 2-thio-N4-methyl-5-hydroxycytidine, 2-thio-N4-methyl-5-methyl-5-azacytidine, 2-thio-N4-methyl-5-amino-5-azacytidine, 2-thio-N4-methyl-5-hydroxy-5-azacytidine, 2-thio-N4-methyl-5-methylpseudoisocytidine, 2-thio-N4-methyl-5-aminopseudoisocytidine, 2-thio-N4-methyl-5-hydroxypseudoisocytidine, 2-thio-N4-amino-5-azacytidine, 2-thio-N4-aminopseudoisocytidine, 2-thio-N4-amino-5-methylcytidine, 2-thio-N4-amino-5-aminocytidine, 2-thio-N4-amino-5-hydroxycytidine, 2-thio-N4-amino-5-methyl-5-azacytidine, 2-thio-N4-amino-5-amino-5-azacytidine, 2-thio-N4-amino-5-hydroxy-5-azacytidine, 2-thio-N4-amino-5-methylpseudoisocytidine, 2-thio-N4-amino-5-aminopseudoisocytidine, 2-thio-N4-amino-5-hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-azacytidine, 2-thio-N4-hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, 2-thio-N4-hydroxy-5-hydroxycytidine, 2-thio-N4-hydroxy-5-methyl-5-azacytidine, 2-thio-N4-hydroxy-5-amino-5-azacytidine, 2-thio-N4-hydroxy-5-hydroxy-5-azacytidine, 2-thio-N4-hydroxy-5-methylpseudoisocytidine, 2-thio-N4-hydroxy-5-aminopseudoisocytidine, 2-thio-N4-hydroxy-5-hydroxypseudoisocytidine, N6-methyladenosine, N6-aminoadenosine, N6-hydroxyadenosine, 7-deazaadenosine, 8-azaadenosine, N6-methyl-7-deazaadenosine, N6-methyl-8-azaadenosine, 7-deaza-8-azaadenosine, N6-methyl-7-deaza-8-azaadenosine, N6-amino-7-deazaadenosine, N6-amino-8-azaadenosine, N6-amino-7-deaza-8-azaadenosine, N6-hydroxyadenosine, N6-hydroxy-7-deazaadenosine, N6-hydroxy-8-azaadenosine, N6-hydroxy-7-deaza-8-azaadenosine, 6-thioguanosine, 7-deazaguanosine, 8-azaguanosine, 6-thio-7-deazaguanosine, 6-thio-8-azaguanosine, 7-deaza-8-azaguanosine, 6-thio-7-deaza-8-azaguanosine, and 5-methoxyuridine.

In some embodiments, the invention relates to one or more non-canonical nucleotides selected from 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-hydroxyuridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, pseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine. In some embodiments, at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% of the non-canonical nucleotides are one or more of 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, pseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine.

In some embodiments, at least about 50%, or at least about 55%%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% of cytidine residues are non-canonical nucleotides selected from 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine.

In some embodiments, at least about 20%, or about 30%, or about 40%, or about 50%, or at least about 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% of uridine residues are non-canonical nucleotides selected from 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, pseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine.

In some embodiments, at least about 10% (e.g. 10%, or about 20%, or about 30%, or about 40%, or about 50%) of guanosine residues are non-canonical nucleotides, and the non-canonical nucleotide is optionally 7-deazaguanosine. In some embodiments, the RNA contains no more than about 50% 7-deazaguanosine in place of guanosine residues.

In some embodiments, the RNA does not contain non-canonical nucleotides in place of adenosine residues.

Note that alternative naming schemes exist for certain non-canonical nucleotides. For example, in certain situations, 5-methylpseudouridine can be referred to as “3-methylpseudouridine” or “N3-methylpseudouridine” or “1-methylpseudouridine” or “N1-methylpseudouridine”.

Nucleotides that contain the prefix “amino” can refer to any nucleotide that contains a nitrogen atom bound to the atom at the stated position of the nucleotide, for example, 5-aminocytidine can refer to 5-aminocytidine, 5-methylaminocytidine, and 5-nitrocytidine. Similarly, nucleotides that contain the prefix “methyl” can refer to any nucleotide that contains a carbon atom bound to the atom at the stated position of the nucleotide, for example, 5-methylcytidine can refer to 5-methylcytidine, 5-ethylcytidine, and 5-hydroxymethylcytidine, nucleotides that contain the prefix “thio” can refer to any nucleotide that contains a sulfur atom bound to the atom at the given position of the nucleotide, and nucleotides that contain the prefix “hydroxy” can refer to any nucleotide that contains an oxygen atom bound to the atom at the given position of the nucleotide, for example, 5-hydroxyuridine can refer to 5-hydroxyuridine and uridine with a methyl group bound to an oxygen atom, wherein the oxygen atom is bound to the atom at the 5C position of the uridine.

Certain embodiments are therefore directed to RNA comprising one or more non-canonical nucleotides, wherein the RNA molecule contains one or more nucleotides that includes one or more substitutions at the 2C and/or 4C and/or 5C positions in the case of a pyrimidine or the 6C and/or 7N and/or 8C positions in the case of a purine. Other embodiments are directed to a therapeutic, wherein the therapeutic contains one or more RNA molecules comprising one or more non-canonical nucleotides, and wherein the one or more RNA molecules comprising one or more non-canonical nucleotides contains one or more nucleotides that includes one or more substitutions at the 2C and/or 4C and/or 5C positions in the case of a pyrimidine or the 6C and/or 7N and/or 8C positions in the case of a purine. In one embodiment, the therapeutic comprises a transfection reagent. In another embodiment, the transfection reagent comprises a cationic lipid, liposome or micelle. In still another embodiment, the liposome or micelle comprises folate and the therapeutic composition has anti-cancer activity. In another embodiment, the one or more nucleotides includes at least one of pseudouridine, 2-thiouridine, 4-thiouridine, 5-azauridine, 5-hydroxyuridine, 5-methyluridine, 5-aminouridine, 2-thiopseudouridine, 4-thiopseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-aminopseudouridine, pseudoisocytidine, N4-methylcytidine, 2-thiocytidine, 5-azacytidine, 5-hydroxycytidine, 5-aminocytidine, 5-methylcytidine, N4-methylpseudoisocytidine, 2-thiopseudoisocytidine, 5-hydroxypseudoisocytidine, 5-aminopseudoisocytidine, 5-methylpseudoisocytidine, 7-deazaadenosine, 7-deazaguanosine, 6-thioguanosine, and 6-thio-7-deazaguanosine. In another embodiment, the one or more nucleotides includes at least one of pseudouridine, 2-thiouridine, 4-thiouridine, 5-azauridine, 5-hydroxyuridine, 5-methyluridine, 5-aminouridine, 2-thiopseudouridine, 4-thiopseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, and 5-aminopseudouridine and at least one of pseudoisocytidine, N4-methylcytidine, 2-thiocytidine, 5-azacytidine, 5-hydroxycytidine, 5-aminocytidine, 5-methylcytidine, N4-methylpseudoisocytidine, 2-thiopseudoisocytidine, 5-hydroxypseudoisocytidine, 5-aminopseudoisocytidine, and 5-methylpseudoisocytidine. In still another embodiment, the one or more nucleotides include at least one of pseudouridine, 2-thiouridine, 4-thiouridine, 5-azauridine, 5-hydroxyuridine, 5-methyluridine, 5-aminouridine, 2-thiopseudouridine, 4-thiopseudouridine, 5-hydroxypseudouridine, and 5-methylpseudouridine, 5-aminopseudouridine and at least one of pseudoisocytidine, N4-methylcytidine, 2-thiocytidine, 5-azacytidine, 5-hydroxycytidine, 5-aminocytidine, 5-methylcytidine, N4-methylpseudoisocytidine, 2-thiopseudoisocytidine, 5-hydroxypseudoisocytidine, 5-aminopseudoisocytidine, and 5-methylpseudoisocytidine and at least one of 7-deazaguanosine, 6-thioguanosine, 6-thio-7-deazaguanosine, and 5-methoxyuridine. In yet another embodiment, the one or more nucleotides includes: 5-methylcytidine and 7-deazaguanosine. In another embodiment, the one or more nucleotides also includes pseudouridine or 4-thiouridine or 5-methyluridine or 5-aminouridine or 4-thiopseudouridine or 5-methylpseudouridine or 5-aminopseudouridine. In a still another embodiment, the one or more nucleotides also includes 7-deazaadenosine. In another embodiment, the one or more nucleotides includes: pseudoisocytidine and 7-deazaguanosine and 4-thiouridine. In yet another embodiment, the one or more nucleotides includes: pseudoisocytidine or 7-deazaguanosine and pseudouridine. In still another embodiment, the one or more nucleotides includes: 5-methyluridine and 5-methylcytidine and 7-deazaguanosine. In a further embodiment, the one or more nucleotides includes: pseudouridine or 5-methylpseudouridine and 5-methylcytidine and 7-deazaguanosine. In another embodiment, the one or more nucleotides includes: pseudoisocytidine and 7-deazaguanosine and pseudouridine. In one embodiment, the RNA comprising one or more non-canonical nucleotides is present in vivo.

Certain non-canonical nucleotides can be incorporated more efficiently than other non-canonical nucleotides into RNA molecules by RNA polymerases that are commonly used for in vitro transcription, due in part to the tendency of these certain non-canonical nucleotides to participate in standard base-pairing interactions and base-stacking interactions, and to interact with the RNA polymerase in a manner similar to that in which the corresponding canonical nucleotide interacts with the RNA polymerase. As a result, certain nucleotide mixtures containing one or more non-canonical nucleotides can be beneficial in part because in vitro-transcription reactions containing these nucleotide mixtures can yield a large quantity of RNA. Certain embodiments are therefore directed to a nucleotide mixture containing one or more nucleotides that includes one or more substitutions at the 2C and/or 4C and/or 5C positions in the case of a pyrimidine or the 6C and/or 7N and/or 8C positions in the case of a purine. Nucleotide mixtures include, but are not limited to (numbers preceding each nucleotide indicate an exemplary fraction of the non-canonical nucleotide triphosphate in an in vitro-transcription reaction, for example, 0.2 pseudoisocytidine refers to a reaction containing adenosine-5′-triphosphate, guanosine-5′-triphosphate, uridine-5′-triphosphate, cytidine-5′-triphosphate, and pseudoisocytidine-5′-triphosphate, wherein pseudoisocytidine-5′-triphosphate is present in the reaction at an amount approximately equal to 0.2 times the total amount of pseudoisocytidine-5′-triphosphate+cytidine-5′-triphosphate that is present in the reaction, with amounts measured either on a molar or mass basis, and wherein more than one number preceding a nucleoside indicates a range of exemplary fractions): 1.0 pseudouridine, 0.1-0.8 2-thiouridine, 0.1-0.8 5-methyluridine, 0.2-1.0 5-hydroxyuridine, 0.2-1.0 5-methoxyuridine, 0.1-1.0 5-aminouridine, 0.1-1.0 4-thiouridine, 0.1-1.0 2-thiopseudouridine, 0.1-1.0 4-thiopseudouridine, 0.1-1.0 5-hydroxypseudouridine, 0.2-1 5-methylpseudouridine, 0.2-1.0 5-methoxypseudouridine, 0.1-1.0 5-aminopseudouridine, 0.2-1.0 2-thiocytidine, 0.1-0.8 pseudoisocytidine, 0.2-1.0 5-methylcytidine, 0.2-1.0 5-hydroxycytidine, 0.2-1.0 5-hydroxymethylcytidine, 0.2-1.0 5-methoxycytidine, 0.1-1.0 5-aminocytidine, 0.2-1.0 N4-methylcytidine, 0.2-1.0 5-methylpseudoisocytidine, 0.2-1.0 5-hydroxypseudoisocytidine, 0.2-1.0 5-aminopseudoisocytidine, 0.2-1.0 N4-methylpseudoisocytidine, 0.2-1.0 2-thiopseudoisocytidine, 0.2-1.0 7-deazaguanosine, 0.2-1.0 6-thioguanosine, 0.2-1.0 6-thio-7-deazaguanosine, 0.2-1.0 8-azaguanosine, 0.2-1.0 7-deaza-8-azaguanosine, 0.2-1.0 6-thio-8-azaguanosine, 0.1-0.5 7-deazaadenosine, and 0.1-0.5 N6-methyladenosine.

In various embodiments, the RNA comprising one or more non-canonical nucleotides composition or synthetic polynucleotide composition (e.g., which may be prepared by in vitro transcription) contains substantially or entirely the canonical nucleotide at positions having adenine or “A” in the genetic code. The term “substantially” in this context refers to at least 90%. In these embodiments, the RNA composition or synthetic polynucleotide composition may further contain (e.g., consist of) 7-deazaguanosine at positions with “G” in the genetic code as well as the corresponding canonical nucleotide “G”, and the canonical and non-canonical nucleotide at positions with G may be in the range of 5:1 to 1:5, or in some embodiments in the range of 2:1 to 1:2. In these embodiments, the RNA composition or synthetic polynucleotide composition may further contain (e.g., consist of) one or more (e.g., two, three or four) of 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine at positions with “C” in the genetic code as well as the canonical nucleotide “C”, and the canonical and non-canonical nucleotide at positions with C may be in the range of 5:1 to 1:5, or in some embodiments in the range of 2:1 to 1:2. In some embodiments, the level of non-canonical nucleotide at positions of “C” are as described in the preceding paragraph. In these embodiments, the RNA composition or synthetic polynucleotide composition may further contain (e.g., consist of) one or more (e.g., two, three, or four) of 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, pseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridineat positions with “U” in the genetic code as well as the canonical nucleotide “U”, and the canonical and non-canonical nucleotide at positions with “U” may be in the range of 5:1 to 1:5, or in some embodiments in the range of 2:1 to 1:2. In some embodiments, the level of non-canonical nucleotide at positions of “U” are as described in the preceding paragraph.

It has now been discovered that combining certain non-canonical nucleotides can be beneficial in part because the contribution of non-canonical nucleotides to lowering the toxicity of RNA molecules can be additive. Certain embodiments are therefore directed to a nucleotide mixture, wherein the nucleotide mixture contains more than one of the non-canonical nucleotides listed above, for example, the nucleotide mixture contains both pseudoisocytidine and 7-deazaguanosine or the nucleotide mixture contains both N4-methylcytidine and 7-deazaguanosine, etc. In one embodiment, the nucleotide mixture contains more than one of the non-canonical nucleotides listed above, and each of the non-canonical nucleotides is present in the mixture at the fraction listed above, for example, the nucleotide mixture contains 0.1-0.8 pseudoisocytidine and 0.2-1.0 7-deazaguanosine or the nucleotide mixture contains 0.2-1.0 N4-methylcytidine and 0.2-1.0 7-deazaguanosine, etc.

In certain situations, for example, when it may not be necessary or desirable to maximize the yield of an in vitro-transcription reaction, nucleotide fractions other than those given above may be used. The exemplary fractions and ranges of fractions listed above relate to nucleotide-triphosphate solutions of typical purity (greater than 90% purity). Larger fractions of these and other nucleotides can be used by using nucleotide-triphosphate solutions of greater purity, for example, greater than about 95% purity or greater than about 98% purity or greater than about 99% purity or greater than about 99.5% purity, which can be achieved, for example, by purifying the nucleotide triphosphate solution using existing chemical-purification technologies such as high-pressure liquid chromatography (HPLC) or by other means. In one embodiment, nucleotides with multiple isomers are purified to enrich the desired isomer.

Other embodiments are directed to a method for inducing a cell in vivo to express a protein of interest by contacting the cell with a RNA molecule that contains one or more non-canonical nucleotides that includes one or more substitutions at the 2C and/or 4C and/or 5C positions in the case of a pyrimidine or the 6C and/or 7N and/or 8C positions in the case of a purine. Still other embodiments are directed to a method for transfecting, reprogramming, and/or gene-editing a cell in vivo by contacting the cell with a RNA molecule that contains one or more non-canonical nucleotides that includes one or more substitutions at the 2C and/or 4C and/or 5C positions in the case of a pyrimidine or the 6C and/or 7N and/or 8C positions in the case of a purine. In one embodiment, the RNA molecule is produced by in vitro transcription. In one embodiment, the RNA molecule encodes one or more reprogramming factors. In another embodiment, the one or more reprogramming factors includes Oct4 protein. In another embodiment, the cell is also contacted with a RNA molecule that encodes Sox2 protein. In yet another embodiment, the cell is also contacted with a RNA molecule that encodes Klf4 protein. In yet another embodiment, the cell is also contacted with a RNA molecule that encodes c-Myc protein. In yet another embodiment, the cell is also contacted with a RNA molecule that encodes Lin28 protein.

Enzymes such as T7 RNA polymerase may preferentially incorporate canonical nucleotides in an in vitro-transcription reaction containing both canonical and non-canonical nucleotides. As a result, an in vitro-transcription reaction containing a certain fraction of a non-canonical nucleotide may yield RNA containing a different, often lower, fraction of the non-canonical nucleotide than the fraction at which the non-canonical nucleotide was present in the reaction. In certain embodiments, references to nucleotide incorporation fractions (for example, “a synthetic RNA molecule containing 50% pseudoisocytidine” or “0.1-0.8 pseudoisocytidine”) therefore can refer both to RNA molecules containing the stated fraction of the nucleotide, and to RNA molecules synthesized in a reaction containing the stated fraction of the nucleotide (or nucleotide derivative, for example, nucleotide-triphosphate), even though such a reaction may yield RNA containing a different fraction of the nucleotide than the fraction at which the non-canonical nucleotide was present in the reaction.

Different nucleotide sequences can encode the same protein by utilizing alternative codons. In certain embodiments, references to nucleotide incorporation fractions therefore can refer both to RNA molecules containing the stated fraction of the nucleotide, and to RNA molecules encoding the same protein as a different RNA molecule, wherein the different RNA molecule contains the stated fraction of the nucleotide.

Certain embodiments are directed to a kit containing one or more materials needed to practice the present invention. In one embodiment, the kit contains one or more synthetic RNA molecules. In one embodiment, the kit contains one or more synthetic RNA molecules that encode one or more reprogramming factors and/or gene-editing proteins. In another embodiment, the one or more synthetic RNA molecules contain one or more non-canonical nucleotides that include one or more substitutions at the 2C and/or 4C and/or 5C positions in the case of a pyrimidine or the 6C and/or 7N and/or 8C positions in the case of a purine. In another embodiment, the kit contains one or more of: a transfection medium, a transfection reagent, a complexation medium, and a matrix solution. In one embodiment, the matrix solution contains fibronectin and/or vitronectin or recombinant fibronectin and/or recombinant vitronectin. In one embodiment, one or more of the components of the kit are present as a plurality of aliquots. In one embodiment, the kit contains aliquots of nucleic acid transfection-reagent complexes. In another embodiment, the kit contains aliquots of nucleic acid transfection-reagent complexes that are provided in a solid form, for example, as frozen or freeze-dried pellets. In yet another embodiment, the kit contains aliquots of medium, wherein each aliquot contains transfection reagent-nucleic acid complexes that are stabilized either by chemical treatment or by freezing.

Transfection, in general, and reprogramming, in particular, can be difficult and time-consuming techniques that can be repetitive and prone to error. However, these techniques are often performed manually due to the lack of automated transfection equipment. Certain embodiments are therefore directed to a system that can transfect, reprogram, and/or gene-edit cells in vivo in an automated or semi-automated manner.

It has now been discovered that the non-canonical nucleotide members of the 5-methylcytidine de-methylation pathway, when incorporated into synthetic RNA, can increase the efficiency with which the synthetic RNA can be translated into protein in vivo, and can decrease the toxicity of the synthetic RNA in vivo. These non-canonical nucleotides include, for example: 5-methylcytidine, 5-hydroxymethylcytidine, 5-formylcytidine, and 5-carboxycytidine (a.k.a. “cytidine-5-carboxylic acid”). Certain embodiments are therefore directed to a nucleic acid. In some embodiments, the nucleic acid is present in vivo. In one embodiment, the nucleic acid is a synthetic RNA molecule. In another embodiment, the nucleic acid comprises one or more non-canonical nucleotides. In one embodiment, the nucleic acid comprises one or more non-canonical nucleotide members of the 5-methylcytidine de-methylation pathway. In another embodiment, the nucleic acid comprises at least one of: 5-methylcytidine, 5-hydroxymethylcytidine, 5-formylcytidine, and 5-carboxycytidine or a derivative thereof. In a further embodiment, the nucleic acid comprises at least one of: pseudouridine, 5-methylpseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-methylcytidine, 5-hydroxymethylcytidine, N4-methylcytidine, N4-acetylcytidine, and 7-deazaguanosine or a derivative thereof. 5-methylcytidine De-Methylation Pathway

Certain embodiments are directed to a protein. Other embodiments are directed to a nucleic acid that encodes a protein. In one embodiment, the protein is a protein of interest. In another embodiment, the protein is selected from: a reprogramming protein and a gene-editing protein. In one embodiment, the nucleic acid is a plasmid. In another embodiment, the nucleic acid is present in a virus or viral vector. In a further embodiment, the virus or viral vector is replication incompetent. In a still further embodiment, the virus or viral vector is replication competent. In one embodiment, the virus or viral vector includes at least one of: an adenovirus, a retrovirus, a lentivirus, a herpes virus, an adeno-associated virus or a natural or engineered variant thereof, and an engineered virus.

It has also been discovered that certain combinations of non-canonical nucleotides can be particularly effective at increasing the efficiency with which synthetic RNA can be translated into protein in vivo, and decreasing the toxicity of synthetic RNA in vivo, for example, the combinations: 5-methyluridine and 5-methylcytidine, 5-hydroxyuridine and 5-methylcytidine, 5-hydroxyuridine and 5-hydroxymethylcytidine, 5-methyluridine and 7-deazaguanosine, 5-methylcytidine and 7-deazaguanosine, 5-methyluridine, 5-methylcytidine, and 7-deazaguanosine, and 5-methyluridine, 5-hydroxymethylcytidine, and 7-deazaguanosine. Certain embodiments are therefore directed to a nucleic acid comprising at least two of: 5-methyluridine, 5-methylcytidine, 5-hydroxymethylcytidine, and 7-deazaguanosine or one or more derivatives thereof. Other embodiments are directed to a nucleic acid comprising at least three of: 5-methyluridine, 5-methylcytidine, 5-hydroxymethylcytidine, and 7-deazaguanosine or one or more derivatives thereof. Other embodiments are directed to a nucleic acid comprising all of: 5-methyluridine, 5-methylcytidine, 5-hydroxymethylcytidine, and 7-deazaguanosine or one or more derivatives thereof. In one embodiment, the nucleic acid comprises one or more 5-methyluridine residues, one or more 5-methylcytidine residues, and one or more 7-deazaguanosine residues or one or more 5-methyluridine residues, one or more 5-hydroxymethylcytidine residues, and one or more 7-deazaguanosine residues.

It has been further discovered that synthetic RNA molecules containing certain fractions of certain non-canonical nucleotides and combinations thereof can exhibit particularly high translation efficiency and low toxicity in vivo. Certain embodiments are therefore directed to a nucleic acid comprising at least one of: one or more uridine residues, one or more cytidine residues, and one or more guanosine residues, and comprising one or more non-canonical nucleotides. In one embodiment, between about 20% and about 80% of the uridine residues are 5-methyluridine residues. In another embodiment, between about 30% and about 50% of the uridine residues are 5-methyluridine residues. In a further embodiment, about 40% of the uridine residues are 5-methyluridine residues. In one embodiment, between about 60% and about 80% of the cytidine residues are 5-methylcytidine residues. In another embodiment, between about 80% and about 100% of the cytidine residues are 5-methylcytidine residues. In a further embodiment, about 100% of the cytidine residues are 5-methylcytidine residues. In a still further embodiment, between about 20% and about 100% of the cytidine residues are 5-hydroxymethylcytidine residues. In one embodiment, between about 20% and about 80% of the guanosine residues are 7-deazaguanosine residues. In another embodiment, between about 40% and about 60% of the guanosine residues are 7-deazaguanosine residues. In a further embodiment, about 50% of the guanosine residues are 7-deazaguanosine residues. In one embodiment, between about 20% and about 80% or between about 30% and about 60% or about 40% of the cytidine residues are N4-methylcytidine and/or N4-acetylcytidine residues. In another embodiment, each cytidine residue is a 5-methylcytidine residue. In a further embodiment, about 100% of the cytidine residues are 5-methylcytidine residues and/or 5-hydroxymethylcytidine residues and/or N4-methylcytidine residues and/or N4-acetylcytidine residues and/or one or more derivatives thereof. In a still further embodiment, about 40% of the uridine residues are 5-methyluridine residues, between about 20% and about 100% of the cytidine residues are N4-methylcytidine and/or N4-acetylcytidine residues, and about 50% of the guanosine residues are 7-deazaguanosine residues. In one embodiment, about 40% of the uridine residues are 5-methyluridine residues and about 100% of the cytidine residues are 5-methylcytidine residues. In another embodiment, about 40% of the uridine residues are 5-methyluridine residues and about 50% of the guanosine residues are 7-deazaguanosine residues. In a further embodiment, about 100% of the cytidine residues are 5-methylcytidine residues and about 50% of the guanosine residues are 7-deazaguanosine residues. In a further embodiment, about 100% of the uridine residues are 5-hydroxyuridine residues. In one embodiment, about 40% of the uridine residues are 5-methyluridine residues, about 100% of the cytidine residues are 5-methylcytidine residues, and about 50% of the guanosine residues are 7-deazaguanosine residues. In another embodiment, about 40% of the uridine residues are 5-methyluridine residues, between about 20% and about 100% of the cytidine residues are 5-hydroxymethylcytidine residues, and about 50% of the guanosine residues are 7-deazaguanosine residues. In some embodiments, less than 100% of the cytidine residues are 5-methylcytidine residues. In other embodiments, less than 100% of the cytidine residues are 5-hydroxymethylcytidine residues. In one embodiment, each uridine residue in the synthetic RNA molecule is a pseudouridine residue or a 5-methylpseudouridine residue. In another embodiment, about 100% of the uridine residues are pseudouridine residues and/or 5-methylpseudouridine residues. In a further embodiment, about 100% of the uridine residues are pseudouridine residues and/or 5-methylpseudouridine residues, about 100% of the cytidine residues are 5-methylcytidine residues, and about 50% of the guanosine residues are 7-deazaguanosine residues.

Other non-canonical nucleotides that can be used in place of or in combination with 5-methyluridine include, but are not limited to: pseudouridine, 5-hydroxyuridine, 5-hydroxypseudouridine, 5-methoxyuridine, 5-methoxypseudouridine, 5-carboxyuridine, 5-carboxypseudouridine, 5-formyluridine, 5-formylpseudouridine, 5-hydroxymethyluridine, 5-hydroxymethylpseudouridine, and 5-methylpseudouridine (a.k.a. “1-methylpseudouridine”, a.k.a. “N1-methylpseudouridine”) or one or more derivatives thereof. Other non-canonical nucleotides that can be used in place of or in combination with 5-methylcytidine and/or 5-hydroxymethylcytidine include, but are not limited to: pseudoisocytidine, 5-methylpseudoisocytidine, 5-hydroxymethylcytidine, 5-formylcytidine, 5-carboxycytidine, 5-methoxycytidine, N4-methylcytidine, N4-acetylcytidine or one or more derivatives thereof. In certain embodiments, for example, when performing only a single transfection, injection or delivery or when the cells, tissue, organ or patient being transfected, injected or delivered to are not particularly sensitive to transfection-associated toxicity or innate-immune signaling, the fractions of non-canonical nucleotides can be reduced. Reducing the fraction of non-canonical nucleotides can be beneficial, in part, because reducing the fraction of non-canonical nucleotides can reduce the cost of the nucleic acid. In certain situations, for example, when minimal immunogenicity of the nucleic acid is desired, the fractions of non-canonical nucleotides can be increased.

Enzymes such as T7 RNA polymerase may preferentially incorporate canonical nucleotides in an in vitro-transcription reaction containing both canonical and non-canonical nucleotides. As a result, an in vitro-transcription reaction containing a certain fraction of a non-canonical nucleotide may yield RNA containing a different, often lower, fraction of the non-canonical nucleotide than the fraction at which the non-canonical nucleotide was present in the reaction. In certain embodiments, references to nucleotide incorporation fractions (for example, “50% 5-methyluridine”) therefore can refer both to nucleic acids containing the stated fraction of the nucleotide, and to nucleic acids synthesized in a reaction containing the stated fraction of the nucleotide (or nucleotide derivative, for example, nucleotide-triphosphate), even though such a reaction may yield a nucleic acid containing a different fraction of the nucleotide than the fraction at which the non-canonical nucleotide was present in the reaction. In addition, different nucleotide sequences can encode the same protein by utilizing alternative codons. In certain embodiments, references to nucleotide incorporation fractions therefore can refer both to nucleic acids containing the stated fraction of the nucleotide, and to nucleic acids encoding the same protein as a different nucleic acid, wherein the different nucleic acid contains the stated fraction of the nucleotide.

Certain embodiments are directed to a nucleic acid comprising a 5′-cap structure selected from Cap 0, Cap 1, Cap 2, and Cap 3 or a derivative thereof. In one embodiment, the nucleic acid comprises one or more UTRs. In another embodiment, the one or more UTRs increase the stability of the nucleic acid. In a further embodiment, the one or more UTRs comprise an alpha-globin or beta-globin 5′-UTR. In a still further embodiment, the one or more UTRs comprise an alpha-globin or beta-globin 3′-UTR. In a still further embodiment, the synthetic RNA molecule comprises an alpha-globin or beta-globin 5′-UTR and an alpha-globin or beta-globin 3′-UTR. In one embodiment, the 5′-UTR comprises a Kozak sequence that is substantially similar to the Kozak consensus sequence. In another embodiment, the nucleic acid comprises a 3′-poly(A) tail. In a further embodiment, the 3′-poly(A) tail is between about 20 nt and about 250 nt or between about 120 nt and about 150 nt long. In a further embodiment, the 3′-poly(A) tail is about 20 nt, or about 30 nt, or about 40 nt, or about 50 nt, or about 60 nt, or about 70 nt, or about 80 nt, or about 90 nt, or about 100 nt, or about 110 nt, or about 120 nt, or about 130 nt, or about 140 nt, or about 150 nt, or about 160 nt, or about 170 nt, or about 180 nt, or about 190 nt, or about 200 nt, or about 210 nt, or about 220 nt, or about 230 nt, or about 240 nt, or about 250 nt long.

Certain embodiments are directed to methods of making nucleic acid drugs, including RNA comprising one or more non-canonical nucleotides. Such methods yield substantially stable RNA.

In various embodiments, the present methods and compositions find use in methods of treating, preventing or ameliorating a disease, disorder and/or condition. For instance, in some embodiments, the described methods of in vivo delivery, including various effective doses, administration strategies and formulations are used in a method of treatment.

In various embodiments, the present methods and compositions find use in methods of altering, modifying and/or changing a tissue (e.g. cosmetically).

In various embodiments, the present methods and compositions include using a nucleic acid drug, including a synthetic RNA, in the diagnosing, treating, preventing or ameliorating of a disease, disorder and/or condition described herein. In various embodiments, the present methods and compositions include using a nucleic acid drug, including a synthetic RNA, in the altering, modifying and/or changing of a tissue (e.g. cosmetically).

Generally speaking, in various embodiments, a synthetic RNA as described herein is administered to a human at specific doses described herein and the synthetic RNA comprises a sequence, sometimes referred to as a target sequence that encodes a protein of interest, which may be a therapeutic protein.

Synthetic RNA comprising only canonical nucleotides can bind to pattern recognition receptors, can be recognized as a pathogen-associated molecular pattern, and can trigger a potent immune response in cells, which can result in translation block, the secretion of inflammatory cytokines, and cell death. It has now been discovered that synthetic RNA comprising certain non-canonical nucleotides can evade detection by the innate immune system, and can be translated at high efficiency into protein, including in humans. It has been further discovered that synthetic RNA comprising at least one of the non-canonical nucleotides described herein, including, for example, a member of the group: 5-methylcytidine, 5-hydroxycytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-methoxyuridine, 5-formyluridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-methoxypseudouridine, and 5-formylpseudouridine can evade detection by the innate immune system, and can be translated at high efficiency into protein, including in humans. Certain embodiments are therefore directed to a method for inducing a cell to express a protein of interest comprising contacting a cell with synthetic RNA. Other embodiments are directed to a method for transfecting a cell with synthetic RNA comprising contacting a cell with a solution comprising one or more synthetic RNA molecules. Still other embodiments are directed to a method for treating a patient comprising administering to the patient synthetic RNA. In one embodiment, the synthetic RNA comprises at least one of the non-canonical nucleotides described herein, including, for example, a member of the group: 5-methylcytidine, 5-hydroxycytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-methoxyuridine, 5-formyluridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-methoxypseudouridine, and 5-formylpseudouridine. In another embodiment, the synthetic RNA encodes a protein of interest. Exemplary RNAs may contain combinations and levels of non-canonical and non-canonical nucleotides as described elsewhere herein, including with respect to the expression of any protein of interest described herein. In yet another embodiment, the method results in the expression of the protein of interest. In a further embodiment, the method results in the expression of the protein of interest in the patient's skin.

Other embodiments are directed to a method for delivering a nucleic acid to a cell in vivo. Still other embodiments are directed to a method for inducing a cell in vivo to express a protein of interest. Still other embodiments are directed to a method for treating a patient. In one embodiment, the method comprises disrupting the stratum corneum. In another embodiment, the method comprises contacting a cell with a nucleic acid. In yet another embodiment, the method results in the cell internalizing the nucleic acid. In a further embodiment, the method results in the cell expressing the protein of interest. In a still further embodiment, the method results in the expression of the protein of interest in the patient. In a still further embodiment, the method results in the amelioration of one or more of the patient's symptoms. In a still further embodiment, the patient is in need of the protein of interest. In a still further embodiment, the patient is deficient in the protein of interest.

Still other embodiments are directed to a method for treating a patient comprising delivering to a patient a composition. In one embodiment, the composition comprises albumin that is treated with an ion-exchange resin or charcoal. In another embodiment, the composition comprises one or more nucleic acid molecules. In yet another embodiment, at least one of the one or more nucleic acid molecules encodes a protein of interest. In one embodiment, the method results in the expression of the protein in the patient's skin. In another embodiment, the method results in the expression of a therapeutically or cosmetically effective amount of the protein of interest in the patient. In yet another embodiment, the method comprises administering a steroid. In a further embodiment, the steroid is a member of the group: hydrocortisone and dexamethasone.

Some embodiments are directed to a therapeutic composition and/or methods of treatment comprising a nucleic acid molecule encoding one or more proteins, wherein at least one of the one or more proteins is an extracellular matrix protein. Still other embodiments are directed to a cosmetic composition comprising a nucleic acid molecule encoding one or more proteins, wherein at least one of the one or more proteins is an extracellular matrix protein.

Pigmentation disorders can cause severe symptoms in patients. It has now been discovered that pigmentation disorders can be treated by delivering to a patient a nucleic acid encoding tyrosinase. Certain embodiments are therefore directed to a method for treating a pigmentation disorder. Other embodiments are directed to a method for altering the pigmentation of a patient. In one embodiment, the method comprises delivering to a patient a nucleic acid encoding tyrosinase. Other embodiments are directed to a cosmetic composition comprising a nucleic acid encoding tyrosinase. Still other embodiments are directed to a therapeutic composition comprising a nucleic acid encoding tyrosinase. Still other embodiments are directed to a method for increasing the ultraviolet absorption of a patient's skin. In one embodiment the method comprises delivering to a patient a nucleic acid encoding tyrosinase. In another embodiment, the method results in an increase in the ultraviolet absorption of the patient's skin. Still other embodiments are directed to a method for reducing photodamage to a person's skin upon exposure to ultraviolet light. In one embodiment, the method results in the reduction of photodamage to the person's skin upon exposure to ultraviolet light. Still other embodiments are directed to a method for treating xeroderma pigmentosum. In one embodiment, the method comprises delivering to a patient a nucleic acid encoding tyrosinase. Still other embodiments are directed to a method for treating epidermolysis bullosa. In one embodiment, the method comprises delivering to a patient a nucleic acid encoding one or more of keratin 5, keratin 14, plectin, an integrin family member, laminin, a laminin subunit, collagen XVII, collagen VII or a biologically active fragment, variant, analogue or family-member thereof. In one embodiment, the method comprises delivering to a patient a nucleic acid encoding collagen type VII. In another embodiment, the method comprises delivering to a patient a nucleic acid encoding melanocortin 1 receptor. Still other embodiments are directed to a method for treating xerosis. In one embodiment, the method comprises delivering to a patient a nucleic acid encoding a hyaluronan synthase. In another embodiment, the patient is diagnosed with atopic dermatitis. In yet another embodiment, the patient is diagnosed with ichthyosis. Certain embodiments are directed to a method for treating a cosmetic condition. Other embodiments are directed to a method for inducing tissue healing. In one embodiment, the method comprises delivering to a patient a nucleic acid encoding a hyaluronan synthase. In another embodiment, the cosmetic condition is a member of the group: wrinkles, sagging skin, thin skin, discoloration, and dry skin. In yet another embodiment, the patient has had cataract surgery. In some embodiments, the nucleic acid is synthetic RNA. In other embodiments, the method results in the amelioration of one or more of the patient's symptoms. Other embodiments are directed to a method for treating an indication by delivering to a cell or a patient a nucleic acid encoding a protein or a peptide. Still other embodiments are directed to a composition comprising a nucleic acid encoding a protein or a peptide. Indications that can be treated using the methods and compositions of the present invention and proteins and peptides that can be encoded by compositions of the present invention are set forth in Table 2A and/or Table 2B, and are given by way of example, and not by way of limitation. In one embodiment, the indication is selected from Table 2A and/or Table 2B. In another embodiment the protein or peptide is selected from Table 2A and/or Table 2B. In yet another embodiment, the indication and the protein or peptide are selected from the same row of Table 2A and/or Table 2B. In a further embodiment, the protein of interest is a member of the group: UCP1, UCP2, and UCP3. Other embodiments are directed to methods for inducing a cell to express a plurality of proteins of interest. In one embodiment, the proteins of interest include at least two members of the group: a lipase, UCP1, UCP2, and UCP3. In another embodiment, the proteins of interest include a lipase and a member of the group: UCP1, UCP2, and UCP3. In another embodiment, the protein is a gene-editing protein. In yet another embodiment, the gene-editing protein targets a gene that is at least partly responsible for a disease phenotype. In yet another embodiment, the gene-editing protein targets a gene that encodes a protein selected from Table 2A and/or Table 2B. In still another embodiment, the gene-editing protein corrects or eliminates, either alone or in combination with one or more other molecules or gene-editing proteins, a mutation that is at least partly responsible for a disease phenotype.

In various embodiments, the present invention contemplates the targeting of the precursor forms and/or mature forms and/or isoforms and/or mutants of any of the proteins disclosed in Table 2A and/or Table 2B and such proteins. In some embodiments, any of the precursor forms and/or mature forms and/or isoforms and/or mutants have enhanced secretion relative to the corresponding wild type proteins. In some embodiments, any of the precursor forms and/or mature forms and/or isoforms and/or mutants have altered half-lives (e.g. serum, plasma, intracellular)—for instance, longer or shorter half-lives. In some embodiments, this is relative to wild type.

TABLE 2A Illustrative Indications Illustrative Indication Illustrative Protein/Peptide Acne Retinol Dehydrogenase 10 Aging Elastin sp|P15502|ELN_HUMAN Elastin (isoform 3) (SEQ ID NO: 486) Aging Collagen Type I P02452|CO1A1_HUMAN Collagen alpha-1(I) chain (SEQ ID NO: 487) P08123|CO1A2_HUMAN Collagen alpha-2(I) chain (SEQ ID NO: 488) Aging Collagen Type III P02461|CO3A1_HUMAN Collagen alpha-1(III) chain (isoform 1) (SEQ ID NO: 489) Aging Collagen Type VII Q02388|CO7A1_HUMAN Collagen alpha-1(VII) chain (SEQ ID NO: 490) Aging Hyaluronan Synthase Aging Telomerase Reverse Transcriptase Albinism Tyrosinase P14679|TYRO_HUMAN Tyrosinase (isoform 1) (SEQ ID NO: 491) Alport Syndrome Collagen Type IV P02462|CO4A1_HUMAN Collagen alpha-1(IV) chain (isoform 1) (SEQ ID NO: 492) P08572|CO4A2_HUMAN Collagen alpha-2(IV) chain (SEQ ID NO: 493) Q01955|CO4A3_HUMAN Collagen alpha-3(IV) chain (isoform 1) (SEQ ID NO: 494) P53420|CO4A4_HUMAN Collagen alpha-4(IV) chain (SEQ ID NO: 495) P29400|CO4A5_HUMAN Collagen alpha-5(IV) chain (isoform 1) (SEQ ID NO: 496) Q14031|CO4A6_HUMAN Collagen alpha-6(IV) (isoform A) (SEQ ID NO: 497) Anemia Erythropoietin Atopic Dermatitis Filaggrin Cutis Laxa Elastin sp|P15502|ELN_HUMAN Elastin (isoform 3) (SEQ ID NO: 486) Dry Skin Filaggrin Dystrophic Epidermolysis Bullosa Collagen Type VII Q02388|CO7A1_HUMAN Collagen alpha-1(VII) chain (SEQ ID NO: 498) Ehlers-Danlos Syndrome Collagen Type V P20908|CO5A1_HUMAN Collagen alpha-1(V) chain (SEQ ID NO: 499) P05997|CO5A2_HUMAN Collagen alpha-2(V) chain (SEQ ID NO: 500) P25940|CO5A3_HUMAN Collagen alpha-3(V) chain (SEQ ID NO: 501) Ehlers-Danlos Syndrome Collagen Type I P02452|CO1A1_HUMAN Collagen alpha-1(I) chain (SEQ ID NO: 487) P08123|CO1A2_HUMAN Collagen alpha-2(I) chain (SEQ ID NO: 488) Epidermolysis bullosa, lethal acantholytic ADAM17 P78536|ADA17_HUMAN Disintegrin and metalloproteinase domain-containing protein 17 (isoform A) (SEQ ID NO: 502) Epidermolysis bullosa, type IV Collagen Type III P02461|CO3A1_HUMAN Collagen alpha-1(III) chain (isoform 1) (SEQ ID NO: 489) Erythropoietic Protoporphyria Ferrochelatase P22830|HEMH_HUMAN Ferrochelatase, mitochondrial (isoform 1) (SEQ ID NO: 503) Eczema Filaggrin Excess Fat Thermogenin P25874|UCP1_HUMAN Mitochondrial brown fat uncoupling protein 1 (SEQ ID NO: 504) Excess Fat Lipase Lipoprotein lipase P06858|LIPL_HUMAN Lipoprotein lipase (SEQ ID NO: 516) Hepatic lipase P11150|LIPC_HUMAN Hepatic triacylglycerol lipase (SEQ ID NO: 517) Pancreatic lipase P16233|LIPP_HUMAN Pancreatic triacylglycerol lipase (SEQ ID NO: 518) Endothelial lipase (isoform 1) Q9Y5X9|LIPE_HUMAN Endothelial lipase (SEQ ID NO: 519) Lysosomal lipase P38571|LICH_HUMAN Lysosomal acid lipase/cholesteryl ester hydrolase (isoform 1) (SEQ ID NO: 520) Hormone sensitive lipase Q05469|LIPS_HUMAN Hormone-sensitive lipas (isoform 1) (SEQ ID NO: 521) Gastric lipase P07098|LIPG_HUMAN Gastric triacylglycerol lipase (isoform 1) (SEQ ID NO: 522) Pancreatic Lipase-Related Protein 1) P54315|LIPR1_HUMAN Inactive pancreatic lipase- related protein 1 (isoform 1) (SEQ ID NO: 523) Pancreatic Lipase-Related Protein 2 P54317|LIPR2_HUMAN Pancreatic lipase-related protein 2 (SEQ ID NO: 524) Carboxyl Ester Lipase P19835|CEL_HUMAN Bile salt-activated lipase (isoform long) (SEQ ID NO: 525) Hypotrichosis ADAM17 P78536|ADA17_HUMAN Disintegrin and metalloproteinase domain-containing protein 17 (isoform A) (SEQ ID NO: 502) Ichthyosis Vulgaris Filaggrin Infections Genetic Antibiotics (e.g. Anti-Sigma Factors) Inflammatory and Bullous Skin Bowel Syndrome Desmoglein 2 Q14126|DSG2_HUMAN Desmoglein-2 (SEQ ID NO: 505) Keratosis Pilaris Retinol Dehydrogenase 10 Oily Skin Retinol Dehydrogenase 10 Osteoarthritis Hyaluronan Synthase Pemphigus Vulgaris Plakophilin-1 Q13835|PKP1_HUMAN Plakophilin-1 (isoform 2) (SEQ ID NO: 506) Pseudoxanthoma elasticum Elastin sp|P15502|ELN_HUMAN Elastin (isoform 3) (SEQ ID NO: 486) Psoriasis Retinol Dehydrogenase 10 Scar Treatment Tyrosinase P14679|TYRO_HUMAN Tyrosinase (isoform 1) (SEQ ID NO: 491) Scarring Elastin sp|P15502|ELN_HUMAN Elastin (isoform 3) (SEQ ID NO: 486) Scarring Collagen Type I P02452|CO1A1_HUMAN Collagen alpha-1(I) chain (SEQ ID NO: 487) P08123|CO1A2_HUMAN Collagen alpha-2(I) chain (SEQ ID NO: 488) Scarring Collagen Type III P02461|CO3A1_HUMAN Collagen alpha-1(III) chain (isoform 1) (SEQ ID NO: 489) Skin Cancer Interferon Interferon, Alpha 1 P01562|IFNA1_HUMAN Interferon alpha-1/13 (SEQ ID NO: 530) Interferon, Alpha 2 P01563|IFNA2_HUMAN Interferon alpha-2 (SEQ ID NO: 531) Interferon, Alpha 4 P05014|IFNA4_HUMAN Interferon alpha-4 (SEQ ID NO: 532) Interferon, Alpha 5 P01569|IFNA5_HUMAN Interferon alpha-5 (SEQ ID NO: 533) Interferon, Alpha 6 P05013|IFNA6_HUMAN Interferon alpha-6 (SEQ ID NO: 534) Interferon, Alpha 7 P01567|IFNA7_HUMAN Interferon alpha-7 (SEQ ID NO: 535) Interferon, Alpha 8 P32881|IFNA8_HUMAN Interferon alpha-8 (SEQ ID NO: 536) Interferon, Alpha 10 P01566|IFN10_HUMAN Interferon alpha-10 (SEQ ID NO: 537) Interferon, Alpha 14 P01570|IFN14_HUMAN Interferon alpha-14 OS (SEQ ID NO: 538) Interferon, Alpha 16 P05015|IFN16_HUMAN Interferon alpha-16 (SEQ ID NO: 539) Interferon, Alpha 17 P01571|IFN17_HUMAN Interferon alpha-17 (SEQ ID NO: 540) Interferon, Alpha 21 P01568|IFN21_HUMAN Interferon alpha-21 (SEQ ID NO: 541) Interferon, Gamma P01579|IFNG_HUMAN Interferon gamma (SEQ ID NO: 542) Interferon, Beta P01574|IFNB_HUMAN Interferon beta (SEQ ID NO: 543) Interferon, Kappa Q9P0W0|IFNK_HUMAN Interferon kappa (SEQ ID NO: 544) Interferon, Epsilon Q86WN2|IFNE_HUMAN Interferon epsilon (SEQ ID NO: 545) Striate Palmoplantar Keratoderma ADAM17 P78536|ADA17_HUMAN Disintegrin and metalloproteinase domain-containing protein 17 (isoform A) (SEQ ID NO: 502) Tanning Tyrosinase P14679|TYRO_HUMAN Tyrosinase (isoform 1) (SEQ ID NO: 491) Vitiligo Melanocyte-Stimulating Hormone Alpha-MSH P01189|138-150 (SEQ ID NO: 526) Beta-MSH P01189|217-234 (SEQ ID NO: 527) Gamma-MSH P01189|77-87 (SEQ ID NO: 528) Proopiomelanocortin P01189|COLI_HUMAN Pro-opiomelanocortin (SEQ ID NO: 529) Vitiligo Tyrosinase P14679|TYRO_HUMAN Tyrosinase (isoform 1) (SEQ ID NO: 491) Warts Interferon Interferon, Alpha 1 P01562|IFNA1_HUMAN Interferon alpha-1/13 (SEQ ID NO: 530) Interferon, Alpha 2 P01563|IFNA2_HUMAN Interferon alpha-2 (SEQ ID NO: 531) Interferon, Alpha 4 P05014|IFNA4_HUMAN Interferon alpha-4 (SEQ ID NO: 532) Interferon, Alpha 5 P01569|IFNA5_HUMAN Interferon alpha-5 (SEQ ID NO: 533) Interferon, Alpha 6 P05013|IFNA6_HUMAN Interferon alpha-6 (SEQ ID NO: 534) Interferon, Alpha 7 P01567|IFNA7_HUMAN Interferon alpha-7 (SEQ ID NO: 535) Interferon, Alpha 8 P32881|IFNA8_HUMAN Interferon alpha-8 (SEQ ID NO: 536) Interferon, Alpha 10 P01566|IFN10_HUMAN Interferon alpha-10 (SEQ ID NO: 537) Interferon, Alpha 14 P01570|IFN14_HUMAN Interferon alpha-14 OS (SEQ ID NO: 538) Interferon, Alpha 16 P05015|IFN16_HUMAN Interferon alpha-16 (SEQ ID NO: 539) Interferon, Alpha 17 P01571|IFN17_HUMAN Interferon alpha-17 (SEQ ID NO: 540) Interferon, Alpha 21 P01568|IFN21_HUMAN Interferon alpha-21 (SEQ ID NO: 541) Interferon, Gamma P01579|IFNG_HUMAN Interferon gamma (SEQ ID NO: 542) Interferon, Beta P01574|IFNB_HUMAN Interferon beta (SEQ ID NO: 543) Interferon, Kappa Q9P0W0|IFNK_HUMAN Interferon kappa (SEQ ID NO: 544) Interferon, Epsilon Q86WN2|IFNE_HUMAN Interferon epsilon (SEQ ID NO: 545) Wound Healing Elastin sp|P15502|ELN_HUMAN Elastin (isoform 3) (SEQ ID NO: 486) Wound Healing Collagen Type I P02452|CO1A1_HUMAN Collagen alpha-1(I) chain (SEQ ID NO: 487) P08123|CO1A2_HUMAN Collagen alpha-2(I) chain (SEQ ID NO: 488) Wound Healing Collagen Type III P02461|CO3A1_HUMAN Collagen alpha-1(III) chain (isoform 1) (SEQ ID NO: 489) Xeroderma Pigmentosum DNA Polymerase Eta Q9Y253|POLH_HUMAN DNA polymerase eta (isoform 1) (SEQ ID NO: 507)

Additional illustrative targets of the present invention include the cosmetic targets listed in Table 6 of International Patent Publication No. WO 2013/151671, the contents of which are hereby incorporated by reference in their entirety.

In various embodiments, the agents of the present invention are used in methods the effect the integumentary system of a human. The present compositions and methods may be used to alter a biological and/or physiological process to, for example, reduce skin sagging, increase skin thickness, increase skin volume, reduce the number of wrinkles, the length of wrinkles and/or the depth of wrinkles, increase skin tightness, firmness, tone and/or elasticity, increase skin hydration and ability to retain moisture, water flow and osmotic balance, increase the levels of skin lipids; increase the extracellular matrix and/or adhesion and communication polypeptides; increase skin energy production; utilization and conservation; improve oxygen utilization; improve skin cell life; improve skin cell immunity defense, heat shock stress response, antioxidant defense capacity to neutralize free radicals, and/or toxic defense; improve the protection and recovery from ultraviolet rays; improve skin cell communication and skin cell innervations; improve cell cohesion/adhesion; improve calcium mineral and other mineral metabolism; improve cell turnover; and improve cell circadian rhythms.

Further still, in some embodiments, the present compositions may be used in the treatment, control, or prevention of a disease, disorder and/or condition and/or may alter, modify or change the appearance of a member of the integumentary system of a subject suffering from a disease, disorder and/or condition such as, but not limited to, acne vulgaris, acne aestivalis, acne conglobata, acne cosmetic, acne fulminans, acne keloidalis nuchae, acne mechanica, acne medicamentosa, acne miliaris necrotica, acne necrotica, acne rosacea, actinic keratosis, acne vulgaris, acne aestivalis, acne conglobata, acne cosmetic, acne fulminans, acne keloidalis nuchae, acne mechanica, acne medicamentosa, acne miliaris necrotica, acne necrotica, acne rosacea, acute urticaria, allergic contact dermatitis, alopecia areata, angioedema, athlete's foot, atopic dermatitis, autoeczematization, baby acne, balding, bastomycosis, blackheads, birthmarks and other skin pigmentation problems, boils, bruises, bug bites and stings, burns, cellulitis, chiggers, chloracne, cholinergic or stress uricara, chronic urticara, cold type urticara, confluent and reticulated papillomatosis, corns, cysts, dandruff, dermatitis herpetiformis, dermatographism, dyshidrotic eczema, diaper rash, dry skin, dyshidrosis, ectodermal dysplasia such as, hyprohidrotic ectodermal dysplasia and X-linked hyprohidrotic ectodermal dysplasia, eczema, epidermaodysplasia verruciformis, erythema nodosum, excoriated acne, exercise-induced anaphylasis folliculitis, excess skin oil, folliculitis, freckles, frostbite, fungal nails, hair density, hair growth rate, halogen acne, hair loss, heat rash, hematoma, herpes simplex infections (e.g. non-genital), hidradenitis suppurativa, hives, hyperhidrosis, hyperpigmentation, hypohidrotic ectodermal dysplasia, hypopigmentation, impetigo, ingrown hair, heat type urticara, ingrown toenail, infantile acne or neonatal acne, itch, irritant contact dermatitis, jock itch, keloid, keratosis pilaris, lichen planus, lichen sclerosus, lupus miliaris disseminatus faciei, melasma, moles, molluscum contagiosum, nail growth rate, nail health, neurodermatitis, nummular eczema, occupational acne, oil acne, onychomycosis, physical urticara, pilonidal cyst, pityriasis rosea, pityriasis versicolor, poison ivy, pomade acne, pseudofolliculitis barbae or acne keloidalis nuchae, psoriasis, psoriatic arthritis, pressure or delayed pressue urticara, puncture wounds such as cuts and scrapes, rash, rare or water type urticara, rhinoplasty, ringworm, rosacea, rothmund-thomson syndrome, sagging of the skin, scabis, scars, seborrhea, seborrheic dermatitis, shingles, skin cancer, skin tag, solar type urticara, spider bite, stretch marks, sunburn, tar acne, tropical acne, thinning of skin, thrush, tinea versicolor, transient acantholytic dermatosis, tycoon's cap or acne necrotica miliaris, uneven skin tone, varicose veins, venous eczema, vibratory angioedema, vitiligo, warts, Weber-Christian disease, wrinkles, x-linked hypohidrotic ectodermal dysplasia, xerotic eczema, yeast infection and general signs of aging.

In some embodiments, there is provided methods of treating, controlling or preventing dry skin with the present compositions. In some embodiments profilaggrin (a protein which is converted to filaggrin) is a protein of interest (e.g. when treating ichthyosis vulgaris).

In some embodiments, there is provided methods of treating, controlling or preventing any one of the various types of psoriasis (e.g. plague psoriasis, guttate psoriasis, pustular psoriasis, inverse psoriasis, and erythrodermic psoriasis). In various embodiments, the protein of interest is any of the products of the genes psoriasis susceptibility 1 through 9 (PSORSI-PSORS9).

Various embodiments relate to the treatment, control, or prevention of eczema (e.g. atopic dermatitis, nummular eczema, dyshidrotic eczema, seborrheic dermatitis, irritant contact dermatitis, allergic contact dermatitis, dyshidrosis, venous eczema, dermatitis herpetiformis, neurodermatitis, autoeczematization and xerotic eczema) and, optionally, one or more of the following may be targeted: filaggrin; three genetic variants, ovo-like 1 (OVOL1), actin-like 9 (ACTL9) and kinesin family member 3 A (KIF3A) have been associated with eczema; and the genes brain-derived neurotrophic factor (BDNF) and tachykinin, precursor 1 (TAC 1).

Hives, or urticaria, including, but not limited to, acute urticaria, chronic urticara and angioedema, physical urticara, pressure or delayed pressue urticara, cholinergic or stress uricara, cold type urticara, heat type urticara, solar type urticara, rare or water type urticara, vibratory angioedema, exercise-induced anaphylasis and dermatographism may be treated with the present compositions by, for example, targeting PLCG-2.

Various embodiments relate to the treatment, control, or prevention of rosacea, which includes, but is not limited to, erthematotelangiectatic rosacea, papulopustular rosacea, phymatous rosacea, and ocular rosacea. Optionally, cathelicidin antimicrobial peptide (CAMP) and/or kallikrein-related peptidase 5 (also known as stratum corneum tryptic enzyme (SCTE)) are proteins of interest.

In some embodiments, there is provided methods of treating, controlling or preventing acne with the present compositions. For example, acne may include, but is not limited to, acneiform eruptions, acne aestivalis, acne conglobata, acne cosmetic, acne fulminans, acne keloidalis nuchae, acne mechanica, acne medicamentosa, acne miliaris necrotica, acne necrotica, acne rosacea, baby acne, blackheads, chloracne, excoriated acne, halogen acne, infantile acne or neonatal acne, lupus miliaris disseminatus faciei, occupational acne, oil acne, pomade acne, tar acne, tropical acne, tycoon's cap or acne necrotica miliaris, pseudofolliculitis barbae or acne keloidalis nuchae, and hidradenitis suppurativa. In these embodiments, the protein of interest may be one or more matrix metalloproteinases (MMP), e.g., matrix metalloproteinase-1 (MMP-1 or interstitial collagenase), matrix metalloproteinase-9 (MMP-9), and matrix metalloproteinase-13 (MMP-13).

In further embodiments, vitiligo is treated with the present compositions, e.g. wherein the NLR family, pyrin domain containing 1 gene (NALP1) gene is targeted.

In some embodiments, the present compositions find use in the treatment, control, or prevention of hyprohidrotic ectodermal dysplasia (HED), e.g. via the ectodysplasin A gene (EDA), receptor (EDAR), and receptor associated death domain (EDARADD).

In some embodiments, the present compositions find use in the treatment, control, or prevention of balding, or hair thinning (e.g. male pattern baldness, or androgenetic alopecia (AGA)) and, optionally, one or more of the following may be the protein of interest: androgen receptor (AR), ectodysplasin A2 receptor (EDA2R) and lysophosphatidic acid receptor 6 (P2RY5).

The present compositions also find use in methods of treatment, control, or prevention of scars and stretch marks (striae), e.g. via collagen, ribosomal s6 kinase, sectrected phosphoprotein 1 (also known as osteopontin), or transforming growth factor beta 3.

Epidermodysplasia verruciformis (also known as Lutz-Lewandowsky epidermodysplasia), a rare autosomal recessive genetic hereditary skin disorder, may also be treated with compositions of the present invention, e.g. by targeted transmembrane channel-like 6 (EVER1) or transmembrane channellike 8 (EVER2) genes.

In some embodiments, skin sagging, thinning or wrinkling may be treated, controlled or prevented with present composition, e.g. by targeting one or more of the proteins of interest such as collagen, elastin, fibroblast growth factor 7, TIMP metallopeptidase inhibitors, matrix metallopeptidases, superoxide dismutase and other extracellular matrix proteins and proteoglycans.

Further embodiments are used in tanning of the skin, such as via melanocyte-stimulating hormone and/or proopiomelanocortin.

In some embodiments, the present compositions may be used for wound treatment. In some embodiments, methods of treating, controlling or preventing wounds with the present compositions comprises additional steps of, for example, cleaning the wound bed to facilitate wound healing and closure, including, but not limited to: debridement, sharp debridement (surgical removal of dead or infected tissue from a wound), optionally including chemical debriding agents, such as enzymes, to remove necrotic tissue; wound dressings to provide the wound with a moist, warm environment and to promote tissue repair and healing (e.g., wound dressings comprising hydrogels (e.g., AQUASORB; DUODERM), hydrocolloids (e.g., AQUACEL; COMFEEL), foams (e.g., LYOFOAM; SPYROSORB), and alginates (e.g., ALGISITE; CURASORB); administration of growth factors to stimulate cell division and proliferation and to promote wound healing e.g. becaplermin; and (iv) soft-tissue wound coverage, a skin graft may be necessary to obtain coverage of clean, non-healing wounds (e.g., autologous skin grafts, cadaveric skin graft, bioengineered skin substitutes (e.g., APLIGRAF; DERMAGRAFT)).

In various embodiments, the nucleic acid drug described herein can be used in a variety of cosmetic/plastic surgery procedures, including, without limitation, a surgical procedure involving skin grafting and an aesthetic or cosmetic surgery (e.g. a facial plastic surgery procedure including, but not limited to blepharoplasty, rhinoplasty, rhytidectomy, genioplasty, facial implants, otoplasty, hair implantation, cleft lip and cleft palate repair, and/or a body plastic surgery procedure including but not limited to abdominoplasty, brachioplasty, thigh lift, breast reduction, breast augmentation, body contouring, liposuction, hand surgery).

In various embodiments, a variety of cancers are treated, controlled or prevented with the present compositions (e.g., colorectal cancer, gallbladder cancer, lung cancer, pancreatic cancer, and stomach cancer). In some embodiments, skin cancer is treated with the present compositions. For instance, the skin cancer is one or more of actinic keratosis, basal cell carcinoma, melanoma, Kaposi's sarcoma, and squamous cell carcinoma. In some embodiments, the present compositions are used adjuvant to complete circumferential peripheral and deep margin assessment, Mohs surgery, radiation (e.g. external beam radiotherapy or brachytherapy), chemotherapy (including but not limited to topical chemotherapy, e.g. with imiquimod or 5-fluorouracil), and cryotherapy. The present compositions also find use in the treatment of various stages of cancers, including skin cancers (e.g. basal cell cancer (BCC), squamous cell cancer (SCC), and melanoma), such as a stage of the American Joint Committee on Cancer (AJCC) TNM system (e.g. one or more of TX, T0, Tis, T1, T1a, T1b, T2, T2A, T2B, T3, T3a, T3b, T4, T4a, T4b, NX, NO, N1, N2, N3, M0, M1a, M1b, M1c) and/or a staging system (e.g. Stage 0, Stage IA, Stage IB, Stage IIA, Stage IIB, Stage IIC, Stage IIIA, Stage IIIB, Stage IIIC, Stage IV).

Illustrative cancers and/or tumors of the present invention include, but are not limited to, a basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.

In various embodiments, one or more rare diseases are treated, controlled or prevented with the present compositions, including, by way of illustration, Erythropoietic Protoporphyria, Hailey-Hailey Disease, Epidermolysis Bullosa (EB), Xeroderma Pigmentosum, Ehlers-Danlos Syndrome, Cutis Laxa, Protein C & Protein S Deficiency, Alport Syndrome, Striate Palmoplantar Keratoderma, Lethal Acantholytic EB, Pseudoxanthoma Elasticum (PXE), Ichthyosis Vulgaris, Pemphigus Vulgaris, and Basal Cell Nevus Syndrome.

In various embodiments, the present compositions are used to treat, control or prevent one or more inflammatory diseases or conditions, such as inflammation, acute inflammation, chronic inflammation, respiratory disease, atherosclerosis, restenosis, asthma, allergic rhinitis, atopic dermatitis, septic shock, rheumatoid arthritis, inflammatory bowel disease, inflammatory pelvic disease, pain, ocular inflammatory disease, celiac disease, Leigh Syndrome, Glycerol Kinase Deficiency, Familial eosinophilia (FE), autosomal recessive spastic ataxia, laryngeal inflammatory disease; Tuberculosis, Chronic cholecystitis, Bronchiectasis, Silicosis and other pneumoconioses.

In various embodiments, the present compositions are used to treat, control or prevent one or more autoimmune diseases or conditions, such as multiple sclerosis, diabetes mellitus, lupus, celiac disease, Crohn's disease, ulcerative colitis, Guillain-Barre syndrome, scleroderms, Goodpasture's syndrome, Wegener's granulomatosis, autoimmune epilepsy, Rasmussen's encephalitis, Primary biliary sclerosis, Sclerosing cholangitis, Autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis, Fibromyalgia, Menier's syndrome; transplantation rejection (e.g., prevention of allograft rejection) pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome, Grave's disease, and other autoimmune diseases.

In various embodiments, the present compositions are used to treat, control or prevent one or more neurologic diseases, including ADHD, AIDS-Neurological Complications, Absence of the Septum Pellucidum, Acquired Epileptiform Aphasia, Acute Disseminated Encephalomyelitis, Adrenoleukodystrophy, Agenesis of the Corpus Callosum, Agnosia, Aicardi Syndrome, Alexander Disease, Alpers' Disease, Alternating Hemiplegia, Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Anencephaly, Aneurysm, Angelman Syndrome, Angiomatosis, Anoxia, Aphasia, Apraxia, Arachnoid Cysts, Arachnoiditis, Arnold-Chiari Malformation, Arteriovenous Malformation, Aspartame, Asperger Syndrome, Ataxia Telangiectasia, Ataxia, Attention Deficit-Hyperactivity Disorder, Autism, Autonomic Dysfunction, Back Pain, Barth Syndrome, Batten Disease, Behcet's Disease, Bell's Palsy, Benign Essential Blepharospasm, Benign Focal Amyotrophy, Benign Intracranial Hypertension, Bernhardt-Roth Syndrome, Binswanger's Disease, Blepharospasm, Bloch-Sulzberger Syndrome, Brachial Plexus Birth Injuries, Brachial Plexus Injuries, Bradbury-Eggleston Syndrome, Brain Aneurysm, Brain Injury, Brain and Spinal Tumors, Brown-Sequard Syndrome, Bulbospinal Muscular Atrophy, Canavan Disease, Carpal Tunnel Syndrome, Causalgia, Cavernomas, Cavernous Angioma, Cavernous Malformation, Central Cervical Cord Syndrome, Central Cord Syndrome, Central Pain Syndrome, Cephalic Disorders, Cerebellar Degeneration, Cerebellar Hypoplasia, Cerebral Aneurysm, Cerebral Arteriosclerosis, Cerebral Atrophy, Cerebral Beriberi, Cerebral Gigantism, Cerebral Hypoxia, Cerebral Palsy, Cerebro-Oculo-Facio-Skeletal Syndrome, Charcot-Marie-Tooth Disorder, Chiari Malformation, Chorea, Choreoacanthocytosis, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), Chronic Orthostatic Intolerance, Chronic Pain, Cockayne Syndrome Type II, Coffin Lowry Syndrome, Coma, including Persistent Vegetative State, Complex Regional Pain Syndrome, Congenital Facial Diplegia, Congenital Myasthenia, Congenital Myopathy, Congenital Vascular Cavernous Malformations, Corticobasal Degeneration, Cranial Arteritis, Craniosynostosis, Creutzfeldt-Jakob Disease, Cumulative Trauma Disorders, Cushing's Syndrome, Cytomegalic Inclusion Body Disease (CIBD), Cytomegalovirus Infection, Dancing Eyes-Dancing Feet Syndrome, Dandy-Walker Syndrome, Dawson Disease, De Morsier's Syndrome, Dejerine-Klumpke Palsy, Dementia—Multi-Infarct, Dementia—Subcortical, Dementia With Lewy Bodies, Dermatomyositis, Developmental Dyspraxia, Devic's Syndrome, Diabetic Neuropathy, Diffuse Sclerosis, Dravet's Syndrome, Dysautonomia, Dysgraphia, Dyslexia, Dysphagia, Dyspraxia, Dystonias, Early Infantile Epileptic Encephalopathy, Empty Sella Syndrome, Encephalitis Lethargica, Encephalitis and Meningitis, Encephaloceles, Encephalopathy, Encephalotrigeminal Angiomatosis, Epilepsy, Erb's Palsy, Erb-Duchenne and Dejerine-Klumpke Palsies, Fabry's Disease, Fahr's Syndrome, Fainting, Familial Dysautonomia, Familial Hemangioma, Familial Idiopathic Basal Ganglia Calcification, Familial Spastic Paralysis, Febrile Seizures (e.g., GEFS and GEFS plus), Fisher Syndrome, Floppy Infant Syndrome, Friedreich's Ataxia, Gaucher's Disease, Gerstmann's Syndrome, Gerstmann-Straussler-Scheinker Disease, Giant Cell Arteritis, Giant Cell Inclusion Disease, Globoid Cell Leukodystrophy, Glossopharyngeal Neuralgia, Guillain-Barre Syndrome, HTLV-1 Associated Myelopathy, Hallervorden-Spatz Disease, Head Injury, Headache, Hemicrania Continua, Hemifacial Spasm, Hemiplegia Alterans, Hereditary Neuropathies, Hereditary Spastic Paraplegia, Heredopathia Atactica Polyneuritiformis, Herpes Zoster Oticus, Herpes Zoster, Hirayama Syndrome, Holoprosencephaly, Huntington's Disease, Hydranencephaly, Hydrocephalus-Normal Pressure, Hydrocephalus, Hydromyelia, Hypercortisolism, Hypersomnia, Hypertonia, Hypotonia, Hypoxia, Immune-Mediated Encephalomyelitis, Inclusion Body Myositis, Incontinentia Pigmenti, Infantile Hypotonia, Infantile Phytanic Acid Storage Disease, Infantile Refsum Disease, Infantile Spasms, Inflammatory Myopathy, Intestinal Lipodystrophy, Intracranial Cysts, Intracranial Hypertension, Isaac's Syndrome, Joubert Syndrome, Kearns-Sayre Syndrome, Kennedy's Disease, Kinsbourne syndrome, Kleine-Levin syndrome, Klippel Feil Syndrome, Klippel-Trenaunay Syndrome (KTS), Klver-Bucy Syndrome, Korsakoffs Amnesic Syndrome, Krabbe Disease, Kugelberg-Welander Disease, Kuru, Lambert-Eaton Myasthenic Syndrome, Landau-Kleffner Syndrome, Lateral Femoral Cutaneous Nerve Entrapment, Lateral Medullary Syndrome, Learning Disabilities, Leigh's Disease, Lennox-Gastaut Syndrome, Lesch-Nyhan Syndrome, Leukodystrophy, Levine-Critchley Syndrome, Lewy Body Dementia, Lissencephaly, Locked-In Syndrome, Lou Gehrig's Disease, Lupus-Neurological Sequelae, Lyme Disease-Neurological Complications, Machado-Joseph Disease, Macrencephaly, Megalencephaly, Melkersson-Rosenthal Syndrome, Meningitis, Menkes Disease, Meralgia Paresthetica, Metachromatic Leukodystrophy, Microcephaly, Migraine, Miller Fisher Syndrome, Mini-Strokes, Mitochondrial Myopathies, Mobius Syndrome, Monomelic Amyotrophy, Motor Neuron Diseases, Moyamoya Disease, Mucolipidoses, Mucopolysaccharidoses, Multi-Infarct Dementia, Multifocal Motor Neuropathy, Multiple Sclerosis, Multiple System Atrophy with Orthostatic Hypotension, Multiple System Atrophy, Muscular Dystrophy, Myasthenia-Congenital, Myasthenia Gravis, Myelinoclastic Diffuse Sclerosis, Myoclonic Encephalopathy of Infants, Myoclonus, Myopathy-Congenital, Myopathy-Thyrotoxic, Myopathy, Myotonia Congenita, Myotonia, Narcolepsy, Neuroacanthocytosis, Neurodegeneration with Brain Iron Accumulation, Neurofibromatosis, Neuroleptic Malignant Syndrome, Neurological Complications of AIDS, Neurological Manifestations of Pompe Disease, Neuromyelitis Optica, Neuromyotonia, Neuronal Ceroid Lipofuscinosis, Neuronal Migration Disorders, Neuropathy-Hereditary, Neurosarcoidosis, Neurotoxicity, Nevus Cavernosus, Niemann-Pick Disease, O'Sullivan-McLeod Syndrome, Occipital Neuralgia, Occult Spinal Dysraphism Sequence, Ohtahara Syndrome, Olivopontocerebellar Atrophy, Opsoclonus Myoclonus, Orthostatic Hypotension, Overuse Syndrome, Pain-Chronic, Paraneoplastic Syndromes, Paresthesia, Parkinson's Disease, Parnyotonia Congenita, Paroxysmal Choreoathetosis, Paroxysmal Hemicrania, Parry-Romberg, Pelizaeus-Merzbacher Disease, Pena Shokeir II Syndrome, Perineural Cysts, Periodic Paralyses, Peripheral Neuropathy, Periventricular Leukomalacia, Persistent Vegetative State, Pervasive Developmental Disorders, Phytanic Acid Storage Disease, Pick's Disease, Piriformis Syndrome, Pituitary Tumors, Polymyositis, Pompe Disease, Porencephaly, Post-Polio Syndrome, Postherpetic Neuralgia, Postinfectious Encephalomyelitis, Postural Hypotension, Postural Orthostatic Tachycardia Syndrome, Postural Tachycardia Syndrome, Primary Lateral Sclerosis, Prion Diseases, Progressive Hemifacial Atrophy, Progressive Locomotor Ataxia, Progressive Multifocal Leukoencephalopathy, Progressive Sclerosing Poliodystrophy, Progressive Supranuclear Palsy, Pseudotumor Cerebri, Pyridoxine Dependent and Pyridoxine Responsive Siezure Disorders, Ramsay Hunt Syndrome Type I, Ramsay Hunt Syndrome Type II, Rasmussen's Encephalitis and other autoimmune epilepsies, Reflex Sympathetic Dystrophy Syndrome, Refsum Disease-Infantile, Refsum Disease, Repetitive Motion Disorders, Repetitive Stress Injuries, Restless Legs Syndrome, Retrovirus-Associated Myelopathy, Rett Syndrome, Reye's Syndrome, Riley-Day Syndrome, SUNCT Headache, Sacral Nerve Root Cysts, Saint Vitus Dance, Salivary Gland Disease, Sandhoff Disease, Schilder's Disease, Schizencephaly, Seizure Disorders, Septo-Optic Dysplasia, Severe Myoclonic Epilepsy of Infancy (SMEI), Shaken Baby Syndrome, Shingles, Shy-Drager Syndrome, Sjogren's Syndrome, Sleep Apnea, Sleeping Sickness, Soto's Syndrome, Spasticity, Spina Bifida, Spinal Cord Infarction, Spinal Cord Injury, Spinal Cord Tumors, Spinal Muscular Atrophy, Spinocerebellar Atrophy, Steele-Richardson-Olszewski Syndrome, Stiff-Person Syndrome, Striatonigral Degeneration, Stroke, Sturge-Weber Syndrome, Subacute Sclerosing Panencephalitis, Subcortical Arteriosclerotic Encephalopathy, Swallowing Disorders, Sydenham Chorea, Syncope, Syphilitic Spinal Sclerosis, Syringohydromyelia, Syringomyelia, Systemic Lupus Erythematosus, Tabes Dorsalis, Tardive Dyskinesia, Tarlov Cysts, Tay-Sachs Disease, Temporal Arteritis, Tethered Spinal Cord Syndrome, Thomsen Disease, Thoracic Outlet Syndrome, Thyrotoxic Myopathy, Tic Douloureux, Todd's Paralysis, Tourette Syndrome, Transient Ischemic Attack, Transmissible Spongiform Encephalopathies, Transverse Myelitis, Traumatic Brain Injury, Tremor, Trigeminal Neuralgia, Tropical Spastic Paraparesis, Tuberous Sclerosis, Vascular Erectile Tumor, Vasculitis including Temporal Arteritis, Von Economo's Disease, Von Hippel-Lindau disease (VHL), Von Recklinghausen's Disease, Wallenberg's Syndrome, Werdnig-Hoffman Disease, Wernicke-Korsakoff Syndrome, West Syndrome, Whipple's Disease, Williams Syndrome, Wilson's Disease, X-Linked Spinal and Bulbar Muscular Atrophy, and Zellweger Syndrome.

In various embodiments, the present compositions are used to treat one or more respiratory diseases, such as asthma, chronic obstructive pulmonary disease (COPD), bronchiectasis, allergic rhinitis, sinusitis, pulmonary vasoconstriction, inflammation, allergies, impeded respiration, respiratory distress syndrome, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, emphysema, Hantavirus pulmonary syndrome (HPS), Loeffler's syndrome, Goodpasture's syndrome, Pleurisy, pneumonitis, pulmonary edema, pulmonary fibrosis, Sarcoidosis, complications associated with respiratory syncitial virus infection, and other respiratory diseases.

In various embodiments, the present compositions are used to treat, control or prevent cardiovascular disease, such as a disease or condition affecting the heart and vasculature, including but not limited to, coronary heart disease (CHD), cerebrovascular disease (CVD), aortic stenosis, peripheral vascular disease, atherosclerosis, arteriosclerosis, myocardial infarction (heart attack), cerebrovascular diseases (stroke), transient ischaemic attacks (TIA), angina (stable and unstable), atrial fibrillation, arrhythmia, vavular disease, and/or congestive heart failure.

In various embodiments, the present compositions are used to treat, control or prevent one or more metabolic-related disorders. In various embodiments, the present invention is useful for the treatment, controlling or prevention of diabetes, including Type 1 and Type 2 diabetes and diabetes associated with obesity. The compositions and methods of the present invention are useful for the treatment or prevention of diabetes-related disorders, including without limitation diabetic nephropathy, hyperglycemia, impaired glucose tolerance, insulin resistance, obesity, lipid disorders, dyslipidemia, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, low HDL levels, high LDL levels, atherosclerosis and its sequelae, vascular restenosis, irritable bowel syndrome, inflammatory bowel disease, including Crohn's disease and ulcerative colitis, other inflammatory conditions, pancreatitis, abdominal obesity, neurodegenerative disease, retinopathy, neoplastic conditions, adipose cell tumors, adipose cell carcinomas, such as liposarcoma, prostate cancer and other cancers, including gastric, breast, bladder and colon cancers, angiogenesis, Alzheimer's disease, psoriasis, high blood pressure, Metabolic Syndrome (e.g. a person has three or more of the following disorders: abdominal obesity, hypertriglyceridemia, low HDL cholesterol, high blood pressure, and high fasting plasma glucose), ovarian hyperandrogenism (polycystic ovary syndrome), and other disorders where insulin resistance is a component, such as sleep apnea.

The compositions and methods of the present invention are useful for the treatment, control, or prevention of obesity, including genetic or environmental, and obesity-related disorders. The obesity-related disorders herein are associated with, caused by, or result from obesity. Examples of obesity-related disorders include obesity, diabetes, overeating, binge eating, and bulimia, hypertension, elevated plasma insulin concentrations and insulin resistance, dyslipidemia, hyperlipidemia, endometrial, breast, prostate, kidney and colon cancer, osteoarthritis, obstructive sleep apnea, gallstones, heart disease, abnormal heart rhythms and arrythmias, myocardial infarction, congestive heart failure, coronary heart disease, sudden death, stroke, polycystic ovary disease, craniopharyngioma, Prader-Willi Syndrome, Frohlich's syndrome, GH-deficient subjects, normal variant short stature, Turner's syndrome, and other pathological conditions showing reduced metabolic activity or a decrease in resting energy expenditure as a percentage of total fat-free mass, e.g, children with acute lymphoblastic leukemia. Further examples of obesity-related disorders are Metabolic Syndrome, insulin resistance syndrome, reproductive hormone abnormalities, sexual and reproductive dysfunction, such as impaired fertility, infertility, hypogonadism in males and hirsutism in females, fetal defects associated with maternal obesity, gastrointestinal motility disorders, such as obesity-related gastro-esophageal reflux, respiratory disorders, such as obesity-hypoventilation syndrome (Pickwickian syndrome), breathlessness, cardiovascular disorders, inflammation, such as systemic inflammation of the vasculature, arteriosclerosis, hypercholesterolemia, lower back pain, gallbladder disease, hyperuricemia, gout, and kidney cancer, and increased anesthetic risk. The compositions and methods of the present invention are also useful to treat Alzheimer's disease.

Nucleic acids, including liposomal formulations containing nucleic acids, when delivered in vivo, can accumulate in the liver and/or spleen. It has now been discovered that nucleic acids encoding proteins can modulate protein expression in the liver and spleen, and that nucleic acids used in this manner can constitute potent therapeutics for the treatment of liver and spleen diseases. Certain embodiments are therefore directed to a method for treating liver and/or spleen disease by delivering to a patient a nucleic acid encoding a protein of interest. Other embodiments are directed to a therapeutic composition comprising a nucleic acid encoding a protein of interest, for the treatment of liver and/or spleen disease. Diseases and conditions of the liver and/or spleen that can be treated include, but are not limited to: hepatitis, alcohol-induced liver disease, drug-induced liver disease, Epstein Barr virus infection, adenovirus infection, cytomegalovirus infection, toxoplasmosis, Rocky Mountain spotted fever, non-alcoholic fatty liver disease, hemochromatosis, Wilson's Disease, Gilbert's Disease, and cancer of the liver and/or spleen.

In some embodiments, the present compositions and methods relate to the treatment of type 1 diabetes, heart disease, including ischemic and dilated cardiomyopathy, macular degeneration, Parkinson's disease, cystic fibrosis, sickle-cell anemia, thalassemia, Fanconi anemia, severe combined immunodeficiency, hereditary sensory neuropathy, xeroderma pigmentosum, Huntington's disease, muscular dystrophy, amyotrophic lateral sclerosis, Alzheimer's disease, cancer, and infectious diseases including hepatitis and HIV/AIDS.

In various embodiments, the present methods and compositions find use in treating or preventing one or more metabolic diseases or disorders. In various embodiments, the present methods and compositions find use in treating or preventing one or more of diseases or disorders of carbohydrate metabolism. diseases or disorders of amino acid metabolism, diseases or disorders of the urea cycle, diseases or disorders of fatty acid metabolism, diseases or disorders of porphyrin metabolism, lysosomal storage disorders, peroxisome biogenesis disorders, and diseases or disorders of purine or pyrimidine metabolism.

In various embodiments, the present methods and compositions find use in treating or preventing one or more of diseases or disorders in the table below. In various embodiments, the present methods and compositions find use in treating or preventing one or more of diseases or disorders in the table below for instance by modulating the genes associated with the diseases in the table below. In some embodiments, the present methods and compositions find use in gene-editing the genes described in the table below using the present compositions.

Category Disease Genes Entrez ID Disorders of Galactosemia GALT, GALK1, GALE 2592, 2584, 2582 carbohydrate Essential fructosuria KHK 3795 metabolism Hereditary fructose ALDOB 229 intolerance Glycogen storage disease G6PC, SLC37A4, SLC17A3 2538, 2542, 10786 type I Glycogen storage disease GAA 2548 type II Glycogen storage disease AGL 178 type III Glycogen storage disease GBE1 2632 type IV Glycogen storage disease PYGM 5837 type V Glycogen storage disease PYGL 5836 type VI Glycogen storage disease PYGM 5837 type VII Glycogen storage disease PHKA1, PHKA2, PHKB, 5255, 5256, 5257, 5260, type IX PHKG1, PHKG2 5261 Glycogen storage disease SLC2A2 6514 type XI Glycogen storage disease ALDOA 226 type XII Glycogen storage disease ENO1, ENO2, ENO3 2023, 2026, 2027 type XIII Glycogen storage disease GYS1, GYS2 2997, 2998 type 0 Pyruvate carboxylase PC 5091 deficiency Pyruvate kinase deficiency PKLR 5313 Transaldolase deficiency TALDO1 6888 Triosephosphate isomerase TPI1 7167 deficiency Fructose bisphosphatase FBP1 2203 deficiency Hyperoxaluria AGXT, GRHPR  189, 9380 Hexokinase deficiency HK1 3098 Glucose-galactose SLC5A1 6523 malabsorption Glucose-6-phosphate G6PD 2539 dehydrogenase deficiency Disorders of Alkaptonuria HGD 3081 amino acid Aspartylglucosaminuria AGA 175 metabolism Methylmalonic acidemia MUT, MCEE, MMAA, 4594, 84693, 166785, MMAB, MMACHC, 326625, 25974, 27249, MMADHC, LMBRD1 55788 Maple syrup urine disease BCKDHA, BCKDHB, DBT, 593, 594, 1629, 1738 DLD Homocystinuria CBS 875 Tyrosinemia FAH, TAT, HPD 2184, 6898, 3242 Trimethylaminuria FMO3 2328 Hartnup disease SLC6A19 340024 Biotinidase deficiency BTD 686 Ornithine OTC 5009 carbamoyltransferase deficiency Carbamoyl-phosphate CPS1 1373 synthase I deficiency disease Citrullinemia ASS, SLC25A13  445, 10165 Hyperargininemia ARG1 383 Hyperhomocysteinemia MTHFR 4524 Hypermethioninemia MAT1A, GNMT, AHCY 4143, 27232, 191 Hyperlysinemias AASS 10157 Nonketotic hyperglycinemia GLDC, AMT, GCSH 2731, 275, 2653 Propionic acidemia PCCA, PCCB 5095, 5096 Hyperprolinemia ALDH4A1, PRODH 8659, 5625 Cystinuria SLC3A1, SLC7A9  6519, 11136 Dicarboxylic aminoaciduria SLC1A1 6505 Glutaric acidemia type 2 ETFA, ETFB, ETFDH 2108, 2109, 2110 Isovaleric acidemia IVD 3712 2-Hydroxyglutaric aciduria L2HGDH, D2HGDH  79944, 728294 Disorders of the N-Acetylglutamate NAGS 162417 urea cycle synthase deficiency Argininosuccinic aciduria ASL 435 Argininemia ARG1 383 Disorders of Very long-chain acyl- ACADVL 37 fatty acid coenzyme A metabolism dehydrogenase deficiency Long-chain 3-hydroxyacyl- HADHA 3030 coenzyme A dehydrogenase deficiency Medium-chain acyl- ACADM 34 coenzyme A dehydrogenase deficiency Short-chain acyl-coenzyme ACADS 35 A dehydrogenase deficiency 3-hydroxyacyl-coenzyme A HADH 3033 dehydrogenase deficiency 2,4 Dienoyl-CoA reductase NADK2 133686 deficiency 3-Hydroxy-3-methylglutaryl- HMGCL 3155 CoA lyase deficiency Malonyl-CoA MLYCD 23417 decarboxylase deficiency Systemic primary carnitine SLC22A5 6584 deficiency Carnitine-acylcarnitine SLC25A20 788 translocase deficiency Carnitine CPT1A 1374 palmitoyltransferase I deficiency Carnitine CPT2 1376 palmitoyltransferase II deficiency Lysosomal acid lipase LIPA 3988 deficiency Gaucher's disease GBA 2629 Disorders of Acute intermittent porphyria HMBS 3145 porphyrin Gunther disease UROS 7390 metabolism Porphyria cutanea tarda UROD 7389 Hepatoerythropoietic UROD 7389 porphyria Hereditary coproporphyria CPOX 1371 Variegate porphyria PPOX 5498 Erythropoietic FECH 2235 protoporphyria Aminolevulinic acid ALAD 210 dehydratase deficiency porphyria Lysosomal Farber disease ASAH1 427 storage Krabbe disease GALC 2581 disorders Galactosialidosis CTSA 5476 Fabry disease GLA 2717 Schindler disease NAGA 4668 GM1 gangliosidosis GLB1 2720 Tay-Sachs disease HEXA 3073 Sandhoff disease HEXB 3074 GM2-gangliosidosis, AB GM2A 2760 variant Niemann-Pick disease SMPD1, NPC1, NPC2 6609, 4864, 10577 Metachromatic ARSA, PSAP  410, 5660 leukodystrophy Multiple sulfatase SUMF1 285362 deficiency Hurler syndrome IDUA 3425 Hunter syndrome IDS 3423 Sanfilippo syndrome SGSH, NAGLU, HGSNAT, 6448, 4669, 138050, 2799 GNS Morquio syndrome GALNS, GLB1 2588, 2720 Maroteaux-Lamy syndrome ARSB 411 Sly syndrome GUSB 2990 Sialidosis NEU1, NEU2, NEU3, NEU4 4758, 4759, 10825, 129807 I-cell disease GNPTAB, GNPTG 79158, 84572 Mucolipidosis type IV MCOLN1 57192 Infantile neuronal ceroid PPT1, PPT2 5538, 9374 lipofuscinosis Jansky-Bielschowsky TPP1 1200 disease Batten disease CLN1, CLN2, CLN3, CLN5, 5538, 1200, 1201, 1203, CLN6, MFSD8, CLN8, CTSD 54982, 256471, 2055, 1509 Kufs disease, Type A CLN6, PPT1 54982, 5538  Kufs disease, Type B DNAJC5, CTSF 80331, 8722  Alpha-mannosidosis MAN2B1, MAN2B2, 4125, 23324, 4123 MAN2C1 Beta-mannosidosis MANBA 4226 Fucosidosis FUCA1 2517 Cystinosis CTNS 1497 Pycnodysostosis CTSK 1513 Salla disease SLC17A5 26503 Infantile free sialic acid SLC17A5 26503 storage disease Danon disease LAMP2 3920 Peroxisome Zellweger syndrome PEX1, PEX2, PEX3, PEX5, 5189, 5828, 8504, 5830, biogenesis PEX6, PEX12, PEX14, 5190, 5193, 5195, 55670 disorders PEX26 Infantile Refsum disease PEX1, PEX2, PEX26 5189, 5828, 55670 Neonatal PEX5, PEX1, PEX10, 5830, 5189, 5192, 5194, adrenoleukodystrophy PEX13, PEX26 55670 RCDP Type 1 PEX7 5191 Pipecolic acidemia PAHX 5264 Acatalasia CAT 847 Hyperoxaluria type 1 AGXT 189 Acyl-CoA oxidase ACOX1 51 deficiency D-bifunctional protein HSD17B4 3295 deficiency Dihydroxyacetonephosphate GNPAT 8443 acyltransferase deficiency X-linked ABCD1 215 adrenoleukodystrophy α-Methylacyl-CoA AMACR 23600 racemase deficiency RCDP Type 2 DHAPAT 8443 RCDP Type 3 AGPS 8540 Adult Refsum disease-1 PHYH 5264 Mulibrey nanism TRIM37 4591 Disorders of Lesch-Nyhan syndrome HPRT 3251 purine or Adenine APRT 353 pyrimidine phosphoribosyltransferase metabolism deficiency Adenosine deaminase ADA 100 deficiency Adenosine monophosphate AMPD1 270 deaminase deficiency type 1 Adenylosuccinate lyase ADSL 158 deficiency Dihydropyrimidine DPYD 1806 dehydrogenase deficiency Miller syndrome DHODH 1723 Orotic aciduria UMPS 7372 Purine nucleoside PNP 4860 phosphorylase deficiency Xanthinuria XDH, MOCS1, MOCS2, 7498, 4337, 4338, 10243 GEPH

The Entrez entries listed in the table above are hereby incorporated by reference in their entireties.

Further, in some embodiments, the present methods and compositions find use in targeting any of the proteins or in treatment of any of the diseases or disorders of Table 2B. In various embodiments, the present invention contemplates the targeting of the full-length and/or truncated forms of any of the proteins disclosed in Table 2B. In various embodiments, the present invention contemplates the targeting of the precursor forms and/or mature forms and/or isoforms of any of the proteins disclosed in Table 2B.

In various embodiments, the present invention contemplates the targeting of a protein having about 60% (e.g. about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%) sequence identity with any of the protein sequences disclosed herein (e.g. in Table 2B).

In various embodiments, the present invention contemplates the targeting of a protein comprising an amino acid sequence having one or more amino acid mutations relative to any of the protein sequences described herein (e.g. in Table 2B). For example, the present invention contemplates the targeting of a protein comprising an amino acid sequence having 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12 amino acid mutations relative to any of the protein sequences described herein (e.g. in Table 2B). In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.

In some embodiments, the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions.

“Conservative substitutions” may be made, for instance, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, lie; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

As used herein, “conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the so modified polypeptide. In addition, glycine and proline may be substituted for one another based on their ability to disrupt α-helices.

As used herein, “non-conservative substitutions” are defined as exchanges of an amino acid by another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.

In various embodiments, the substitutions may also include non-classical amino acids (e.g. selenocysteine, pyrrolysine, N-formylmethionine β-alanine, GABA and δ-Aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β methyl amino acids, C α-methyl amino acids, N α-methyl amino acids, and amino acid analogs in general).

In Table 2B, all Illustrative Identifiers (e.g. Gene Seq nos. and references are hereby incorporated by reference in their entireties).

TABLE 2B Illustrative Proteins, Illustrative Peptides, and Illustrative Indications Protein/Peptide Illustrative Identifier Reference Description/Illustrative Indication(s) Transthyretin (TTR) his gene encodes transthyretin, one of the three prealbumins including alpha-1- (SEQ ID NOs: 637 and 638) antitrypsin, transthyretin and orosomucoid. Transthyretin is a carrier protein; it Gene ID: 7276 transports thyroid hormones in the plasma and cerebrospinal fluid, and also transports retinol (vitamin A) in the plasma. The protein consists of a tetramer of identical subunits. More than 80 different mutations in this gene have been reported; most mutations are related to anyloid deposition, affecting predominantly peripheral nerve and/or the heart, and a small portion of the gene mutations is non- amyloidogenic. The diseases caused by mutations include amyloidotic polyneuropathy, euthyroid hyperthyroxinaemia, amyloidotic vitreous opacities, cardiomyopathy, oculoleptomeningeal amyloidosis, meningocerebrovascular amyloidosis, carpal tunnel syndrome, etc. Endothelial Cell Specific This gene encodes a secreted protein which is mainly expressed in the endothelial Molecule 1 cells in human lung and kidney tissues. The expression of this gene is regulated by (SEQ ID NO: 629 to 632) cytokines, suggesting that it may play a role in endothelium-dependent pathological Gene ID: 11082 disorders. The transcript contains multiple polyadenylation and mRNA instability signals. Two transcript variants encoding different isoforms have been found for this gene Parathyroid hormone PTH is secreted by the chief cells of the parathyroid glands. PTH plays an important P01270|PTHY_HUMAN role in the regulation of serum calcium, serum phosphate, and vitamin D synthesis. Parathyroid hormone (SEQ ID NO: 508) BMP-1 GeneSeq BMP1 belongs to the transforming growth factor-beta (TGFB) super- family. Bone Accession P80618 morphogenic proteins induce cartilage and bone formation, play important role in WO8800205 nephrogesis, and play an important role in the development of many organs, P13497/BMP1_HUMAN including lung, heart, teeth, gut, skin, and particularly the kidney. BMP-1 activity can Bone morphogenetic be determined using the following assays known in the art: Nat Genet. 2001 January; protein 1 27(1): 84-8; Eur J Biochem 1996 Apr. 1; 237(1): 295-302; J Biol Chem, Vol. 274, (isoform BMP1-3) Issue 16, 10897-10902, Apr. 16, 1999; and Hogan, B. L. M. (1996) Genes Dev. 10, (SEQ ID NO: 169) 1580-1594. Induction of Cartilage, Tissue and Bone Growth, and Diabetes P13497-2|BMP1_HUMAN Isoform BMP1-1 of Bone morphogenetic protein 1 (isoform BMP1-1) (SEQ ID NO: 509) P13497-3|BMP1_HUMAN Isoform BMP1-4 of Bone morphogenetic protein 1 (isoform BMP1-4) (SEQ ID NO: 510) P13497-4|BMP1_HUMAN Isoform BMP1-5 of Bone morphogenetic protein 1 (isoform BMP1-5) (SEQ ID NO: 511) P13497-5|BMP1_HUMAN Isoform BMP1-6 of Bone morphogenetic protein 1 (isoform BMP1-6) (SEQ ID NO: 512) P13497-6|BMP1_HUMAN Isoform BMP1-7 of Bone morphogenetic protein 1 (isoform BMP1-7) (SEQ ID NO: 513) BMP-2 GeneSeq BMP-2 belongs to the transforming growth factor-beta (TGFB) superfamily. Bone Accession P80619 morphogenic protein induces bone formation. BMP-2 activity can be determined WO8800205 using the following assays known in the art: Nat Genet. 2001 January; 27(1): 84-8; Eur J P12643/BMP2_HUMAN Biochem 1996 Apr. 1; 237(1): 295-302; J Biol Chem, Vol. 274, Issue 16, 10897-10902, Bone morphogenetic Apr. 16, 1999; and Hogan, B. L. M. (1996) Genes Dev. 10, 1580-1594. protein 2 Induction of Cartilage, Tissue and Bone Growth, and Diabetes. Infarction recovery. (SEQ ID NO: 170) Bone repair. Osteoporosis. BMP-3 BMP-3 belongs to the transforming growth factor-beta (TGFB) superfamily. Bone P12645|BMP3_HUMAN morphogenic protein induces bone formation. BMP-3 activity can be determined Bone morphogenetic using the following assays known in the art: Nat Genet. 2001 January; 27(1): 84-8; Eur J protein 3 Biochem 1996 Apr. 1; 237(1): 295-302; J Biol Chem, Vol. 274, Issue 16, 10897-10902, (SEQ ID NO: 514) Apr. 16, 1999; and Hogan, B. L. M. (1996) Genes Dev. 10, 1580-1594. Induction of Cartilage, Tissue and Bone Growth, and Diabetes BMP-2B GeneSeq BMP-2b belongs to the transforming growth factor-beta (TGFB) superfamily. Bone Accession W24850 U.S. morphogenic protein induces bone formation. BMP-2b activity can be determined Pat. No. 5,631,142 using the following assays known in the art: Nat Genet. 2001 January; 27(1): 84-8; Eur J P12644/BMP4_HUMAN Biochem 1996 Apr. 1; 237(1): 295-302; I Biol Cbcre, Vol. 274, Issue 16, 10897-10902, Bone morphogenetic Apr. 16, 1999; and Hogan, B. L. M. (1996) Genes Dev. 10, 1580-1594. Induction of protein 4 Cartilage, Tissue and Bone Growth, and Diabetes (SEQ ID NO: 171) BMP-4 GeneSeq BMP-4 belongs to the transforming growth factor-beta (TGFB) superfamily. Bone Accession B02796 morphogenic protein induces bone formation. BMP-4 activity can be determined WO0020591 using the following assays known in the art: Nat Genet. 2001 January; 27(1): 84-8; Eur J P12644/BMP4_HUMAN Biochem 1996 Apr. 1; 237(1): 295-302; J Biol Chem, Vol. 274, Issue 16, 10897-10902, Bone morphogenetic Apr. 16, 1999; and Hogan, B. L. M. (1996) Genes Dev. 10, 1580-1594. protein 4 Induction of Cartilage, Tissue and Bone Growth, and Diabetes (SEQ ID NO: 172) BMP-5 GeneSeq BMP-5 belongs to the transforming growth factor-beta (TGFB) superfamily. Bone Accession B02797 morphogenic protein induces bone formation. BMP-5 activity can be determined WO0020591 using the following assays known in the art: Nat Genet. 2001 January; 27(1): 84-8; Eur J P22003/BMP5_HUMAN Biochem 1996 Apr. 1; 237(1): 295-302; J Biol Chem, Vol. 274, Issue 16, 10897-10902, Bone morphogenetic Apr. 16, 1999; and Hogan, B. L. M. (1996) Genes Dev. 10, 1580-1594. protein 5 Induction of Cartilage, Tissue and Bone Growth, and Diabetes (isoform 1) (SEQ ID NO: 173) P22003-2|BMP5_HUMAN Isoform 2 of Bone morphogenetic protein 5 (isoform 2) (SEQ ID NO: 515) BMP-6 GeneSeq BMP-6 belongs to the transforming growth factor-beta (TGFB) superfamily. Bone Accession R32904 U.S. morphogenic protein induces bone formation. BMP-6 activity can be determined Pat. No. 5,187,076 using the following assays known in the art: Nat Genet. 2001 January; 27(1): 84-8; Eur J P22004/BMP6_HUMAN Biochem 1996 Apr. 1; 237(1): 295-302; J Biol Chem, Vol. 274, Issue 16, 10897-10902, Bone morphogenetic Apr. 16, 1999; and Hogan, B. L. M. (1996) Genes Dev. 10, 1580-1594. protein 6 Induction of Cartilage, Tissue and Bone Growth, and Diabetes. Hemochromatosis. (SEQ ID NO: 174) Osteogenic Protein-1; OP- OP-1 belongs to the transforming growth factor-beta (TGFB) superfamily. Bone 1; BMP-7 GeneSeq morphogenic protein induces bone formation. OP-1 activity can be determined using Accession W34783 the following assays known in the art: Nat Genet. 2001 January; 27(1): 84-8; Eur J WO973462 Biochem 1996 Apr. 1; 237(1): 295-302; J Biol Chem, Vol. 274, Issue 16, 10897-10902, P18075/BMP7_HUMAN Apr. 16, 1999; and Hogan, B. L. M. (1996) Genes Dev. 10, 1580-1594. Bone morphogenetic Induction of Cartilage, Tissue and Bone Growth, and Diabetes protein 7 (SEQ ID NO: 175) BMP7 Variant A OP-1 belongs to the transforming growth factor-beta (TGFB) superfamily. Bone (SEQ ID NO: 579) morphogenic protein induces bone formation. OP-1 activity can be determined using the following assays known in the art: Nat Genet. 2001 January; 27(1): 84-8; Eur J Biochem 1996 Apr. 1; 237(1): 295-302; J Biol Chem, Vol. 274, Issue 16, 10897-10902, Apr. 16, 1999; and Hogan, B. L. M. (1996) Genes Dev. 10, 1580-1594. Induction of Cartilage, Tissue and Bone Growth, and Diabetes BMP7 Variant B OP-1 belongs to the transforming growth factor-beta (TGFB) superfamily. Bone (SEQ ID NO: 580) morphogenic protein induces bone formation. OP-1 activity can be determined using the following assays known in the art: Nat Genet. 2001 January; 27(1): 84-8; Eur J Biochem 1996 Apr. 1; 237(1): 295-302; J Biol Chem, Vol. 274, Issue 16, 10897-10902, Apr. 16, 1999; and Hogan, B. L. M. (1996) Genes Dev. 10, 1580-1594. Induction of Cartilage, Tissue and Bone Growth, and Diabetes BMP7 Variant C OP-1 belongs to the transforming growth factor-beta (TGFB) superfamily. Bone (SEQ ID NO: 581) morphogenic protein induces bone formation. OP-1 activity can be determined using the following assays known in the art: Nat Genet. 2001 January; 27(1): 84-8; Eur J Biochem 1996 Apr. 1; 237(1): 295-302; J Biol Chem, Vol. 274, Issue 16, 10897-10902, Apr. 16, 1999; and Hogan, B. L. M. (1996) Genes Dev. 10, 1580-1594. Induction of Cartilage, Tissue and Bone Growth, and Diabetes Osteogenic Protein-2 OP-2 belongs to the transforming growth factor-beta (TGFB) superfamily. Bone GeneSeq Accession morphogenic protein induces bone formation. OP-2 activity can be determined using R57973 WO9406399 the following assays known in the art: Nat Genet. 2001 January; 27(1): 84-8; Eur J P34820/BMP8B_HUMAN Biochem 1996 Apr. 1; 237(1): 295-302; J Biol Chem, Vol. 274, Issue 16, 10897-10902, Bone morphogenetic Apr. 16, 1999; and Hogan, B. L. M. (1996) Genes Dev. 0, 1580-1594. protein 8B Induction of Cartilage, Tissue and Bone Growth, and Diabetes (SEQ ID NO: 176) GDF-1 GeneSeq Members of the TGF-beta family of proteins initiate cell signaling by binding to Accession R60961 heteromeric receptor complexes of type I (TbetaRI) and type II (TbetaRII) WO9406449 serine/threonine kinase receptors (reviewed by Massague, J. et al. (1994) Trends P27539/GDF1_HUMAN Cell Biol. 4: 172 178; Miyazono, K. et al., (1994) Adv. Immunol. 55: 181-220). Embryonic Activation of this heteromeric receptor complex occurs when TGF-beta binds to growth/differentiation TbetaRII, which then recruits and phosphorylates TbetaRI. Activated TbetaRI then factor 1 propagates the signal to downstream targets (Chen, F. and Weinberg, R. A. (1995) (SEQ ID NO: 177) PNA892: 1565-1569; Wrana, J. L. et al. (1994) Nature 370: 341 347). The effect of GDF-1 on signaling can be assayed by treating Primary BAECs transferred with a construct called p3TP-Lux, containing a TGF-beta responsive promoter fused to a reporter gene, and measuring luciferase gene expression (Wrana et al., 1994, Nature 370: 341-347). Developmental disorders, Induction of Cartilage, Tissue and Bone Growth, and Diabetes BMP-9 GeneSeq BMP-9 belongs to the transforming growth factor-beta (TGFB) superfamily. Bone Accession R86903 morphogenic protein induces bone formation. BMP-9 activity can be determined WO9533830 using the following assays known in the art: Nat Genet. 2001 January; 27(1): 84-8; Eur J Q9UK05/GDF2_HUMAN Biochem 1996 Apr. 1; 237(1): 295-302; J Biol Chem, Vol. 274, Issue 16, 10897-10902, Growth/differentiation Apr. 16, 1999; and Hogan, B. L. M. (1996) Genes Dev. 10, 1580-1594. factor 2 Induction of Cartilage, Tissue and Bone Growth, and Diabetes (SEQ ID NO: 178) BMP-10 GeneSeq BMP-10 belongs to the transforming growth factor-beta (TGFB) superfamily. Bone Accession R66202 morphogenic protein induces bone formation. BMP-10 activity can be determined WO9426893 using the following assays known in the art: Nat Genet. 2001 January; 27(1): 84-8; Eur J O95393/BMP10_HUMAN Biochem 1996 Apr. 1; 237(1): 295-302; J Biol Chem, Vol. 274, Issue 16, 10897-10902, Bone morphogenetic Apr. 16, 1999; and Hogan, B. L. M. (1996) Genes Dev. 10, 1580-1594. protein 10 Induction of Cartilage, Tissue and Bone Growth, and Diabetes (SEQ ID NO: 179) BMP-12 GeneSeq BMP-12 belongs to the transforming growth factor-beta (TGFB) superfamily. Bone Accession R78734 morphogenic protein induces bone formation. BMP-12 activity can be determined WO9516035 using the following assays known in the art: Nat Genet. 2001 January; 27(1): 84-8; Eur J Q7Z4P5/GDF7_HUMAN Biochem 1996 Apr. 1; 237(1): 295-302; J Biol Chem, Vol. 274, Issue 16, 10897-10902, Growth/differentiation Apr. 16, 1999; and Hogan, B. L. M. (1996) Genes Dev. 10, 1580-1594. factor 7 Induction of Cartilage, Tissue and Bone Growth, and Diabetes (SEQ ID NO: 180) BMP-15 GeneSeq BMP-15 belongs to the transforming growth factor-beta (TGFB) superfamily. Bone Accession W11261 morphogenic protein induces bone formation. BMP-15 activity can be determined W09636710 using the following assays known in the art: Nat Genet. 2001 January; 27(1): 84-8; Eur J O95972/BMP15_HUMAN Biochem 1996 Apr. 1; 237(1): 295-302; J Biol Chem, Vol. 274, Issue 16, 10897-10902, Bone morphogenetic Apr. 16, 1999; and Hogan, B. L. M. (1996) Genes Dev. 10, 1580-1594. protein 15 Induction of Cartilage, Tissue and Bone Growth, and Diabetes (SEQ ID NO: 181) BMP-17 GeneSeq BMP-17 belongs to the transforming growth factor-beta (TGFB) superfamily. Bone Accession Y17870 morphogenic protein induces bone formation. BMP-17 activity can be determined WO9929718 using the following assays known in the art: Nat Genet. 2001 January; 27(1): 84-8; Eur J SEQ ID NO: 2 from U.S. Biochem 1996 Apr. 1; 237(1): 295-302; J Biol Chem, Vol. 274, Issue 16, 10897-10902, Pat. No. 7,151,086 Apr. 16, 1999; and Hogan, B. L. M. (1996) Genes Dev. 10, 1580-1594. (SEQ ID NO: 182) Induction of Cartilage, Tissue and Bone Growth, and Diabetes BMP-18 GeneSeq BMP-18 belongs to the transforming growth factor-beta (TGFB) superfamily. Bone Accession Y17871 morphogenic protein induces bone formation. BMP-18 activity can be determined WO9929718 using the following assays known in the art: Nat Genet. 2001 January; 27(1): 84-8; Eur J SEQ ID NO: 4 from U.S. Biochem 1996 Apr. 1; 237(1): 295-302; J Biol Chem, Vol. 274, Issue 16, 10897-10902, Pat. No. 7,151,086 Apr. 16, 1999; and Hogan, B. L. M. (1996) Genes Dev. 10, 1580-1594. (SEQ ID NO: 183) Induction of Cartilage, Tissue and Bone Growth, and Diabetes Inhibin alpha GeneSeq The inhibin beta A subunit joins the alpha subunit to form a pituitary FSH secretion Accession B02806 inhibitor. Inhibin has been shown to regulate gonadal stromal cell proliferation WO0020591 negatively and to have tumor-suppressor activity. In addition, serum levels of inhibin P05111/INHA_HUMAN have been shown to reflect the size of granulosa-cell tumors and can therefore be Inhibin alpha chain used as a marker for primary as well as recurrent disease. Tumor suppressor activity (SEQ ID NO: 184) of inhibin can be determined using assays known in the art: Matzuk et al., Nature 1992 Nov. 26: 360 (6402); 313-9. Tumor suppression. Inhibin beta GeneSeq The inhibin beta A subunit joins the alpha subunit to form a pituitary FSH secretion Accession H02808 inhibitor. Inhibin has been shown to regulate gonadal stromal cell proliferation WO0020591 negatively and to have tumour-suppressor activity. In addition, serum levels of inhibin P08476/INHBA_HUMAN have been shown to reflect the size of granulosa-cell tumors and can therefore be Inhibin beta A chain used as a marker for primary as well as recurrent disease. Tumor suppressor activity (SEQ ID NO: 185) of inhibin can be determined using assays known in the art: Matzuk et al., Nature P09529/INHBB_HUMAN 1992 Nov. 26: 360 (6402); 313-9. Tumor suppression. Inhibin beta B chain (SEQ ID NO: 186) Cerberus Protein Cerebus is believed to be involved in the inhibition of BMP activity BMP activity, in the GeneSeq Accession presence of the antagonist Cerebus, can be determined using the following assays W86032 WO9849296 known in the art: Nat Genet. 2001 January; 27(1): 84-8; Eur J Biochem 1996 Apr. 1; O95813/CER1_HUMAN 237(1): 295-302; J Biol Chem, Vol. 274, Issue 16, 10897-10902, Apr. 16, 1999; and Cerberus Hogan, B. L. M. (1996) Genes Dev. 10, 1580-1594. BMP Antagonist useful for (SEQ ID NO: 187) Osteosarcoma, abnormal bone growth. Soluble BMP Receptor Soluble BMP receptor kinase protein-3 is involved in the binding of BMPs. Soluble Kinase Protein-3 BMP receptor kinase protein-3 is useful as an antagonist for the inhibition of BMP GeneSeq Accession activity. BMP activity, in the presence of the soluble antagonist BMP receptor kinase R95227 WO9614579 protein-3, can be determined using the following assays known in the art: Nat Genet. Q13873/BMPR2_HUMAN 2001 January; 27(1): 84-8; Eur J Biochem 1996 Apr. 1; 237(1): 295-302; J Biol Chem, Bone morphogenetic Vol. 274, Issue 16, 10897-10902, Apr. 16, 1999; and Hogan, B. L. M. (1996) Genes protein receptor type-2 Dev. 10, 1580-1594. BMP Antagonist useful for Osteosarcoma, abnormal bone (SEQ ID NO: 188) growth. BMP Processing Enzyme BMPs belong to the transforming growth factor-beta (TGFB) superfamily. Bone Furin GeneSeq Accession morphogenic protein induces bone formation. BMP activity, in the presence of the W36099 WO9741250 Furin, can be determined using the following assays known in the art: Nat Genet. P09958/FURIN_HUMAN 2001 January; 27(1): 84-8; Eur J Biochem 1996 Apr. 1; 237(1): 295-302; J Biol Chem, Vol. Furin 274, Issue 16, 10897-10902, Apr. 16, 1999; and Hogan, B. L. M. (1996) Genes Dev. (SEQ ID NO: 189) 10, 1580-1594. Bone formation or Regeneration Abnormalities TGF-beta 1 GeneSeq Members of the TGF-beta family of proteins initiate cell signaling by binding to Accession R29657 heteromeric receptor complexes of type I (TbetaRI) and type II (TbetaRII) WO9216228 serine/threonine kinase receptors (reviewed by Massague, J. et al. (1994) Trends P01137/TGFB1_HUMAN Cell Biol. 4: 172 178; Miyazono, K. et al. (1994) Adv. Immunol. 55: 181-220). Transforming growth Activation of this heteromeric receptor complex occurs when TGF-beta. binds to factor beta-1 TbetaRII, which then recruits and phosphorylates TbetaRI. Activated TbetaRI then (SEQ ID NO: 190) propagates the signal to downstream targets (Chen, F. and Weinberg. R. A. (1995) PNA892: 1565-1569; Wrana, J. L. et al. (1994) Nature 370: 341. The effect of TGF betas on signaling can be assayed by treating Primary BAECs transfected with a construct called p3TP-Lux, containing a TGF- beta responsive promoter fused to a reporter gene, and measuring luciferase gene expression (Wrana et al., 1994, Nature 370: 341-347). Useful for treating cancer and to promote wound healing. TGF-beta 2 GeneSeq Members of the TGF-beta family of proteins initiate cell signaling by binding to Accession R39659 heteromeric receptor complexes of type I (TbetaRI) and type II (TbetaRII) EP542679 serine/threonine kinase receptors (reviewed by Massague, J. et al. (1994) Trends P61812/TGFB2_HUMAN Cell Biol. 4: 172 178; Miyazono, K. et al. (1994) Adv. Immunol. 55: 181-220). Transforming growth Activation of this heteromeric receptor complex occurs when TGF-beta. binds to factor beta-2 TbetaRII, which then recruits and phosphorylates TbetaRI. Activated TbetaRI then (SEQ ID NO: 191) propagates the signal to downstream targets (Chen, F. and Weinberg. R. A. (1995) PNA892: 1565-1569; Wrana, J. L. et al. (1994) Nature 370: 341. The effect of TGF betas on signaling can be assayed by treating Primary BAECs transfected with a construct called p3TP-Lux, containing a TGF- beta responsive promoter fused to a reporter gene, and measuring luciferase gene expression (Wrana et al., 1994, Nature 370: 341-347). Useful for treating cancer and to promote wound healing. ZTGF-beta 9 GeneSeq Members of the TGF-beta family of proteins initiate cell signaling by binding to Accession Y70654 heteromeric receptor complexes of type I (TbetaRI) and type II (TbetaRII) WO0015798 serine/threonine kinase receptors (reviewed by Massague, J. et al. (1994) Trends SEQ ID NO: 2 of Cell Biol. 4: 172 178; Miyazono, K. et al. (1994) Adv. Immunol. 55: 181-220). WO0015798 Activation of this heteromeric receptor complex occurs when TGF-beta. binds to (SEQ ID NO: 192) TbetaRII, which then recruits and phosphorylates TbetaRI. Activated TbetaRI then propagates the signal to downstream targets (Chen, F. and Weinberg. R. A. (1995) PNA892: 1565-1569; Wrana, J. L. et al. (1994) Nature 370: 341. The effect of TGF betas on signaling can be assayed by treating Primary BAECs transfected with a construct called p3TP-Lux, containing a TGF- beta responsive promoter fused to a reporter gene, and measuring luciferase gene expression (Wrana et al., 1994, Nature 370: 341-347). Useful for treating cancer and to promote wound healing. Anti-TGF beta family Members of the TGF-beta family of proteins initiate cell signaling by binding to antibodies GB2305921 heteromeric receptor complexes of type I (TbetaRI) and type II (TbetaRII) serine/threonine kinase receptors (reviewed by Massague, J. et al. (1994) Trends Cell Biol. 4: 172 178; Miyazono, K. et al. (1994) Adv. Immunol. 55: 181-220). Activation of this heteromeric receptor complex occurs when TGF-beta. binds to TbetaRII, which then recruits and phosphorylates TbetaRI. Activated TbetaRI then propagates the signal to downstream targets (Chen, F. and Weinberg. R. A. (1995) PNA892: 1565-1569; Wrana, J. L. et al. (1994) Nature 370: 341. The effect of TGF betas on signaling in the presence of an anti-TGF beta antibody, can be assayed by treating Primary BAECs transfected with a construct called p3TP-Lux, containing a TGF-beta responsive promoter fused to a reporter gene, and measuring luciferase gene expression (Wrana et al., 1994, Nature 370: 341-347). Useful for control of fibrosis, immune, and inflammatory disease. Latent TGF beta binding Members of the TGF-beta family of proteins initiate cell signaling by binding to protein II GeneSeq heteromeric receptor complexes of type I (TbetaRI) and type II (TbetaRII) Accession Y70552 serine/threonine kinase receptors (reviewed by Massague, J. et al. (1994) Trends WO0012551 Cell Biol. 4: 172 178; Miyazono, K. et al. (1994) Adv. Immunol. 55: 181-220). Q14767/LTBP2_HUMAN Activation of this heteromeric receptor complex occurs when TGF-beta. binds to Latent-transforming TbetaRII, which then recruits and phosphorylates TbetaRI. Activated TbetaRI then growth factor beta-binding propagates the signal to downstream targets (Chen, F. and Weinberg. R. A. (1995) protein 2 PNA892: 1565-1569; Wrana, J. L. et al. (1994) Nature 370: 341. The effect of TGF (SEQ ID NO: 193) betas on signaling in the presence of a TGF beta binding protein, can be assayed by treating Primary BAECs transfected with a construct called p3TP-Lux, containing a TGF-beta responsivepromoter fused to a reporter gene, and measuring luciferase gene expression (Wrana et al., 1994, Nature 370: 341-347). Useful for inhibiting tissue or tumor growth. MP52 GeneSeq Members of the TGF-beta family of proteins initiate cell signaling by binding to Accession W36100 heteromeric receptor complexes of type I (TbetaRI) and type II (TbetaRII) WO9741250 serine/threonine kinase receptors (reviewed by Massague, J. et al. (1994) Trends P43026/GDF5_HUMAN Cell Biol. 4: 172 178; Miyazono, K. et al. (1994) Adv. Immunol. 55: 181-220). Growth/differentiation Activation of this heteromeric receptor complex occurs when TGF-beta. binds to factor 5 TbetaRII, which then recruits and phosphorylates TbetaRI. Activated TbetaRI then (SEQ ID NO: 194) propagates the signal to downstream targets (Chen, F. and Weinberg. R. A. (1995) PNA892: 1565-1569; Wrana, J. L. et al. (1994) Nature 370: 341. The effect of TGF betas on signaling can be assayed by treating Primary BAECs transfected with a construct called p3TP-Lux, containing a TGF- beta responsive promoter fused to a reporter gene, and measuring luciferase gene expression (Wrana et al., 1994, Nature 370: 341-347). Bone formation or Regeneration Abnormalities b57 Protein GeneSeq BMPs are involved in the induction of bone formation. Specific antagonists are useful Accession W69293 is preventing this activity from occurring. BMP activity, in the presence of b57 protein, WO9837195 can be determined using the following assays known in the art: Nat Genet. 2001 January; SEQ ID NO: 2 of 27(1): 84-8; Eur J Biochem 1996 Apr. 1; 237(1): 295-302; J Biol Chem, Vol. 274, Issue WO9837195 16, 1089-10902, Apr. 16, 1999; and Hogan, B. L. M. (1996) Genes Deve. 10, 1580-1594. (SEQ ID NO: 195) BMP Antagonist useful for Osteosarcoma, abnormal bone growth. Resistin GeneSeq This gene belongs to the family defined by mouse FIZZI and FIZZ3/Resistin genes. Accession W69293 The characteristic feature of this family is the C-terminal stretch of 10 cys residues WO0064920 with identical spacing. The mouse homolog of this protein is secreted by adipocytes, Q9HD89/RETN_HUMAN may be the hormone potentially linking obesity to type II diabetes. Ability of resistin to Resistin influence type II diabetes can be determined using assays known in the art: Pontoglio (isoform 1) et al., J Clin Invest 1998 May 15; 101(10): 2215-22. Type II diabetes and Syndrome (SEQ ID NO: 196) X. Galectin-4 GeneSeq Galectins are a family of carbohydrate-binding proteins characterized by an affinity for Accession W11841 beta- galactoside containing glycoconjugates. Ability of Galectin-4 polypeptides to WO9703190 bind lactose can be determined using assays known in the art: Wada, et al., J Biol P56470/LEG4_HUMAN Chem 1997 Feb. 28; 272(9): 6078-86. Lactose intolerance. Galectin-4 (SEQ ID NO: 197) APM-I; ACRP- 30; ACPR30 gene is exclusively expressed in adipose tissue. ACRP30 is thought to Famoxin GeneSeq increase fatty acid oxidation by muscle tissue. Ability of ACRP30 polypeptides to Accession Y71035 influence obesity and fat oxidation can be determined using assays known in the art: W00026363 Fruebis et al., Proc Nat'l Acad Sci USA 2001 Feb. 13; 98(4): 2005-10. Obesity, Q15848/ADIPO_HUMAN Metabolic disorders, Lipid Metabolism; Hormone Secretion. Adiponectin (SEQ ID NO: 198) ACRP-30 Homologue; ACPR30 gene is exclusively expressed in adipose tissue. ACRP30 is thought to Complement Component increase fatty acid oxidation by muscle tissue. Ability of ACRP30 homologue Clq C GeneSeq Accession polypeptides to influence obesity and fat oxidation can be determined using assays B30234 WO0063376 known in the art: Fruebis et al., Proc Nat'l Acad Sci USA 2001 Feb. 13; 98(4): 2005-10. P02747/C1QC_HUMAN Obesity, Metabolic disorders, Lipid Metabolism; Hormone Secretion. Complement C1q subcomponent subunit C (SEQ ID NO: 199) Calpain-10a GeneSeq Calpain is believed to play a role in insulin secretion and insulin activity, and therefore Accession Y79567 may be useful in the treatment of type II diabetes. Ability of Calpain-10 to influence WO0023603 type II diabetes can be determined using assays known in the art: Pontoglio et al., J Q9HC96/CAN10_HUMAN Clin Invest 1998 May 15; 101(10): 2215-22. Diabetes mellitus; Regulation of Insulin Calpain-10 secretory response; Insulin mediated glucose transport disorders. (Isoform A) (SEQ ID NO: 200) Calpain-10b GeneSeq Calpain is believed to play a role in insulin secretion and insulin activity, and therefore Accession Y79568 may be useful in the treatment of type II diabetes. Ability of Calpain-10 to influence WO0023603 type II diabetes can be determined using assays known in the art: Pontoglio et al., J Q9HC96- Clin Invest 1998 May 15; 101(10): 2215-22. Diabetes mellitus; Regulation of Insulin 2/CAN10_HUMAN Isoform secretory response; Insulin mediated glucose transport disorders. B of Calpain-10 (SEQ ID NO: 201) Calpain-10c GeneSeq Calpain is believed to play a role in insulin secretion and insulin activity, and therefore Accession Y79569 may be useful in the treatment of type II diabetes. Ability of Calpain-10 to influence WO0023603 type II diabetes can be determined using assays known in the art: Pontoglio et al., J Q9HC96- Clin Invest 1998 May 15; 101(10): 2215-22. Diabetes mellitus; Regulation of Insulin 3/CAN10_HUMAN Isoform secretory response; Insulin mediated glucose transport disorders. C of Calpain-10 (SEQ ID NO: 202) PDGF-D GeneSeq Vascular Endothelial Growth Factor. Proliferation assay using NR6R-3T3 cells Accession Y71130 (Rizzino 1988 Cancer Res. 48: 4266). Wound Healing; Atherosclermis. WO0027879 Q9GZP0/PDGFD_HUMAN Platelet-derived growth factor D (isoform 1) (SEQ ID NO: 203) FasL GeneSeq Accession Activities associated with apoptosis and immune system functions. Activity can be Y28594 WO9936079 determined using Apoptosis assays known in the art: Walczak et al. (1996) EMBOJ P48023/TNFL6_HUMAN 16: 5386-5397. Apoptosis-related disorders; Auto- immune disorders; Graft v-Host Tumor necrosis factor disorders. ligand superfamily member 6 (isoform 1) (SEQ ID NO: 204) Chondro modulin-like Chondromodulin proteins are cartilage proteins thought to confer resistance to protein GeneSeq anglogeneis, and thus are useful as anti-angiogenic agents that may have utility in Accession Y71262 combating cancer. Ability of Chondromodulin-like protein to inhibit vascularization can WO0029579 be determined using assays known in the art: Hirakie et al., J Biol Chem 1997 Dec. SEQ ID NO: 2 from 19; 272(51): 32419-26. Antianglogenic agent; Osteoblast proliferation stimulator; WO0029579 prevents vascularization of cartilage tissue; Useful to treat cancer. (SEQ ID NO: 370) Patched GeneSeq Patched is a tumour-suppressor receptor for Sonic hedgehog (shh), which is a protein Accession W72969 U.S. that controls developmental patterning and growth. Ability of soluble Patched to bind Pat. No. 5,837,538 to and inhibit the activities of shh can be determined using assays known in the art: Q13635/PTC1_HUMAN Stone et al., Nature 1996 Nov. 14; 384(6605): 129-34. Receptor for Hedgehog cellular Protein patched homolog 1 proliferation signaling molecule. This receptor is useful as a means of preventing (isoform L) cellular proliferation via the shh signaling path- way, thus useful for cancers. (SEQ ID NO: 205) Patched-2 GeneSeq Patched is a tumour-suppressor receptor for Sonic hedgehog (shh), which is a protein Accession Y43261 that controls developmental patterning and growth. Ability of soluble Patched to bind WO9953058 to and inhibit the activities of shh can be determined using assays known in the art: Q9Y6C5/PTC2_HUMAN Stone et al., Nature 1996 Nov. 14; 384(6605): 129-34. Receptor for Hedgehog cellular Protein patched homolog 2 proliferation signaling molecule. This receptor is useful as a means of preventing (isoform 1) cellular proliferation via the shh signaling path- way, thus useful for cancers. (SEQ ID NO: 206) Maspin; Protease Inhibitor Maspin is a member of the serpin family of serine protease inhibitors that is thought to 5 GeneSeq Accession suppress tumor metastasis. The inhibitory effects of Maspin and other protease R50938 WO9405804 inhibitors can be assayed using methods known in the art such as a labeled protease P36952/SPB5_HUMAN substrate, for example, Universal Protease Substrate (casein, resorufin-labeled): Serpin B5 Roche Molecular Biochemicals, Cat. No. 1080733. Tumor suppressor which is down- (isoform 1) regulated in breast cancers. The maspin protein has tumour suppressing and (SEQ ID NO: 207) invasion sup- pressing activity. Endostatin GeneSeq Endostatin is believed to inhibit effects of capillary endothelial cell proliferation. The Accession B28399 inhibitory effects of endostatin can be assayed using assays disclosed by Cao et al. WO0064946 (1996) J. Biol. Chem. 271 29461-29467. Anti-angiogenic activity. Useful in the P39060/COIA1_HUMAN prevention and/or treatment of cancers. Collagen alpha-1(XVIII) chain (isoform 1) (SEQ ID NO: 208) aFGF; FGF-1 GeneSeq Fibroblast Growth Factor Proliferation assay using NR6R-3T3 cells (Rizzino 1988 Accession P94037 Cancer Res. 48: 4266); Examples 23 and 39 disclosed herein. Promotion of growth EP298723 and proliferation of cells, such as epithelial cells and keratinocytes. Antagonists may P05230/FGF1_HUMAN be useful as anti-cancer agents. Diabetes, Metabolic Disease, Obesity. Fibroblast growth factor 1 (isoform 1) (SEQ ID NO: 209) bFGF; FGF-2 GeneSeq Fibroblast Growth Factor Proliferation assay using NR6R-3T3 cells (Rizzino 1988 Accession R06685 Cancer Res. 48: 4266); Examples 23 and 39 disclosed herein. Promotion of growth FR2642086 and proliferation of cells, such as epithelial cells and keratinocytes. Antagonists may P09038/FGF2_HUMAN be useful as anti-cancer agents. Fibroblast growth factor 2 (isoform 1) (SEQ ID NO: 210) FGF-3; INT-2 GeneSeq Fibroblast Growth Factor Proliferation assay using NR6R-3T3 cells (Rizzino 1988 Accession R07824 Cancer Res. 48: 4266); Examples 23 and 39 disclosed herein. Promotion of growth WO9503831 and proliferation of cells, such as epithelial cells and keratinocytes. Antagonists may P11487/FGF3_HUMAN be useful as anti-cancer agents. Fibroblast growth factor 3 (SEQ ID NO: 211) FGF-4; HST-1; HBGF-4 Fibroblast Growth Factor Proliferation assay using NR6R-3T3 cells (Rizzino 1988 GeneSeq Accession Cancer Res. 48: 4266); Examples 23 and 39 disclosed herein. Promotion of growth R07825 WO9503831 and proliferation of cells, such as epithelial cells and keratinocytes. Antagonists may P08620/FGF4_HUMAN be useful as anti-cancer agents. Fibroblast growth factor 4 (isoform 1) (SEQ ID NO: 212) FGF-5 GeneSeq Fibroblast Growth Factor Proliferation assay using NR6R-3T3 cells (Rizzino 1988 Accession W22600 Cancer Res. 48: 4266); Examples 23 and 39 disclosed herein. Promotion of growth WO9730155 and proliferation of cells, such as epithelial cells and keratinocytes. Antagonists may P12034/FGF5_HUMAN be useful as anti-cancer agents. Fibroblast growth factor 5 (isoform long) (SEQ ID NO: 213) FGF-6; Heparin binding Fibroblast Growth Factor Proliferation assay using NR6R-3T3 cells (Rizzino 1988 secreted transforming Cancer Res. 48: 4266); Examples 23 and 39 disclosed herein. Promotion of growth factor-2 GeneSeq and proliferation of cells, such as epithelial cells and keratinocytes. Antagonists may Accession R58555 be useful as anti-cancer agents. EP613946 P10767/FGF6_HUMAN Fibroblast growth factor 6 (SEQ ID NO: 214) FGF-8 GeneSeq Fibroblast Growth Factor Proliferation assay using NR6R-3T3 cells (Rizzino 1988 Accession R80783 Cancer Res. 48: 4266); Examples 23 and 39 disclosed herein. Promotion of growth WO9524928 and proliferation of cells, such as epithelial cells and keratinocytes. Antagonists may P55075/FGF8_HUMAN be useful as anti-cancer agents. Fibroblast growth factor 8 (isoform 8E) (SEQ ID NO: 215) FGF-9; Gila activating Fibroblast Growth Factor Proliferation assay using NR6R-3T3 cells (Rizzino 1988 factor GeneSeq Accession Cancer Res. 48: 4266); Examples 23 and 39 disclosed herein. Promotion of growth R70822 WO9503831 and proliferation of cells, such as epithelial cells and keratinocytes. Hair growth. P31371/FGF9_HUMAN Antagonists may be useful as anti-cancer agents. Fibroblast growth factor 9 (SEQ ID NO: 216) FGF-12; Fibroblast growth Fibroblast Growth Factor Proliferation assay using NR6R-3T3 cells (Rizzino 1988 factor homologous factor-1 Cancer Res. 48: 4266); Examples 23 and 39 disclosed herein. Promotion of growth GeneSeq Accession and proliferation of cells, such as epithelial cells and keratinocytes. Antagonists may W06309 WO9635708 be useful as anti-cancer agents. P61328/FGF12_HUMAN Fibroblast growth factor 12 (isoform 1) (SEQ ID NO: 217) FGF-19 GeneSeq Fibroblast Growth Factor Proliferation assay using NR6R-3T3 cells (Rizzino 1988 Accession Y08582 Cancer Res. 48: 4266); Examples 23 and 39 disclosed herein. Promotion of growth WO9927100 and proliferation of cells, such as epithelial cells and keratinocytes. Chronic liver O95750/FGF19_HUMAN disease. Primary biliary cirrhosis. Bile acid-induced liver damage. Antagonists may be Fibroblast growth factor 19 useful as anti-cancer agents. (SEQ ID NO: 218) FGF-16 GeneSeq Fibroblast Growth Factor Proliferation assay using NR6R-3T3 cells (Rizzino 1988 Accession Y05474 Cancer Res. 48: 4266); Examples 23 and 39 disclosed herein. Promotion of growth WO9918128 and proliferation of cells, such as epithelial cells and keratinocytes. Antagonists may O43320/FGF16_HUMAN be useful as anti-cancer agents. Fibroblast growth factor 16 (SEQ ID NO: 219) FGF-18 GeneSeq Fibroblast Growth Factor Proliferation assay using NR6R-3T3 cells (Rizzino 1988 Accession Y08590 Cancer Res. 48: 4266); Examples 23 and 39 disclosed herein. Promotion of growth WO9927100 and proliferation of cells, such as epithelial cells and keratinocytes. Antagonists may O76093/FGF18_HUMAN be useful as anti-cancer agents. Fibroblast growth factor 18 (SEQ ID NO: 220) flt-3 ligand GeneSeq Stem Cell Progenitor Chemokine activities can be determined using assays known in Accession R67541 the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited EP627487 by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, P49771|FLT3L_HUMAN NJ. Promotion of immune cell growth and/or differentiation. Fms-related tyrosine kinase 3 ligand (isoform 1) (SEQ ID NO: 221) VEGF-110 GeneSeq Promotes the growth and/or proliferation of endothelial cells. VEGF activity can be Accession Y69417 determined using assays known in the art, such as those disclosed in International WO0013702 Publication No. WO0045835, for example. Promotion of growth and proliferation of SEQ ID NO: 11 from cells, such as vascular endothelial cells. Antagonists may be useful as anti- WO0013702 angiogenic agents, and may be applicable for cancer. (SEQ ID NO: 222) VEGF-121 GeneSeq Promotes the growth and/or proliferation of endothelial cells. VEGF activity can be Accession B50432 determined using assays known in the art, such as those disclosed in International WO0071713 Publication No. WO0045835, for example. Promotion of growth and proliferation of SEQ ID NO: 2 from cells, such as vascular endothelial cells. Antagonists may be useful as anti- WO0071713 angiogenic agents, and may be applicable for cancer. (SEQ ID NO: 223) VEGF-138 GeneSeq Promotes the growth and/or proliferation of endothelial cells. VEGF activity can be Accession Y43483 determined using assays known in the art, such as those disclosed in International WO9940197 Publication No. WO0045835, for example. Promotion of growth and proliferation of SEQ ID NO: 4 of cells, such as vascular endothelial cells. Antagonists may be useful as anti- WO99/40197 angiogenic agents, and may be applicable for cancer. (SEQ ID NO: 371) VEGF-145 GeneSeq Promotes the growth and/or proliferation of endothelial cells. VEGF activity can be Accession Y69413 determined using assays known in the art, such as those disclosed in International WO0013702 Publication No. WO0045835, for example. Promotion of growth and proliferation of SEQ ID NO: 4 from cells, such as vascular endothelial cells. Antagonists may be useful as anti- WO0013702 angiogenic agents, and may be applicable for cancer. (SEQ ID NO: 224) VEGF-162 GeneSeq Promotes the growth and/or proliferation of endothelial cells. VEGF activity can be Accession Y43484 determined using assays known in the art, such as those disclosed in International W09940197 Publication No. WO0045835, for example. Promotion of growth and proliferation of SEQ ID NO: 8 of cells, such as vascular endothelial cells. Antagonists may be useful as anti- WO99/40197 angiogenic agents, and may be applicable for cancer. (SEQ ID NO: 372) VEGF-165 GeneSeq Promotes the growth and/or proliferation of endothelial cells. VEGF activity can be Accession Y69414 determined using assays known in the art, such as those disclosed in International WO0013702 Publication No. WO0045835, for example. Promotion of growth and proliferation of SEQ ID NO: 6 from cells, such as vascular endothelial cells. Antagonists may be useful as anti- WO0013702 angiogenic agents, and may be applicable for cancer. (SEQ ID NO: 225) VEGF-182 GeneSeq Promotes the growth and/or proliferation of endothelial cells. VEGF activity can be Accession Y43483 determined using assays known in the art, such as those disclosed in International W09940197 Publication No. WO0045835, for example. Promotion of growth and proliferation of SEQ ID NO: 6 of cells, such as vascular endothelial cells. Antagonists may be useful as anti- WO99/40197 angiogenic agents, and may be applicable for cancer. (SEQ ID NO: 373) VEGF-189 GeneSeq Promotes the growth and/or proliferation of endothelial cells. VEGF activity can be Accession Y69415 determined using assays known in the art, such as those disclosed in International WO0013702 Publication No. WO0045835, for example. Promotion of growth and proliferation of SEQ ID NO: 8 from cells, such as vascular endothelial cells. Antagonists may be useful as anti- WO0013702 angiogenic agents, and may be applicable for cancer. (SEQ ID NO: 226) VEGF-206 GeneSeq Promotes the growth and/or proliferation of endothelial cells. VEGF activity can be Accession Y69416 determined using assays known in the art, such as those disclosed in International W00013702 Publication No. WO0045835, for example. Promotion of growth and proliferation of SEQ ID NO: 10 from cells, such as vascular endothelial cells. Antagonists may be useful as anti- WO0013702 angiogenic agents, and may be applicable for cancer. (SEQ ID NO: 227) VEGF-D GeneSeq Promotes the growth and/or proliferation of endothelial cells. VEGF activity can be Accession W53240 determined using assays known in the art, such as those disclosed in International WO9807832 Publication No. WO0045835, for example. Promotion of growth and proliferation of O43915/VEGFD_HUMAN cells, such as vascular endothelial cells. Antagonists may be useful as anti- Vascular endothelial angiogenic agents, and may be applicable for cancer. growth factor D (SEQ ID NO: 374) VEGF-E; VEGF-X Promotes the growth and/or proliferation of endothelial cells. VEGF activity can be GeneSeq Accession determined using assays known in the art, such as those disclosed in International Y33679 WO9947677 Publication No. WO0045835, for example. Promotion of growth and proliferation of SEQ ID NO: 2 from cells, such as vascular endothelial cells. Antagonists may be useful as anti- WO9947677 angiogenic agents, and may be applicable for cancer. (SEQ ID NO: 228) VEGF Receptor; KDR; flk- Receptor for VEGF polypeptides VEGF activity, in the presence of flk-1 polypeptides, 1 GeneSeq Accession can be determined using assays known in the art, such as those disclosed in W69679 WO9831794 International Publication No. WO0045835, for example. VEGF Receptor. Fusion P35968/VGFR2_HUMAN protein with the extracellular domain is useful as an anti-angiogenic agent. Vascular endothelial Antagonists may be useful in the promotion of angiogenesis. growth factor receptor 2 (isoform 1) (SEQ ID NO: 229) Soluble VEGF Receptor Receptor for VEGF polypeptides VEGF activity, in the presence of VEGF Receptor GeneSeq Accession polypeptides, can be determined using assays known in the art, such as those W47037 U.S. Pat. No. disclosed in International Publication No. WO0045835, for example. VEGF Receptor. 5,712,380 Fusion protein with the extracellular domain is useful as an anti-angiogenic agent. sVEGF-RI (FIG. 3) of Antagonists may be useful in the promotion of angiogenesis. U.S. Pat. No. 5,712,380 (SEQ ID NO: 442) sVEGF-RII (FIG. 11) of U.S. Pat. No. 5,712,380 (SEQ ID NO: 443) sVEGF-RTMI (FIG. 15) of U.S. Pat. No. 5,712,380 (SEQ ID NO: 444) sVEGF-RTMII (FIG. 13) of U.S. Pat. No. 5,712,380 (SEQ ID NO: 445) flt-1 GeneSeq Accession Receptor for VEGF polypeptides VEGF activity, in the presence of flt-1 polypeptides, Y70751 WO0021560 can be determined using assays known in the art, such as those disclosed in P17948/VGFR1_HUMAN International Publication No. WO0045835, for example. VEGF Receptor. Fusion Vascular endothelial protein with the extracellular domain is useful as an anti-angiogenic agent. growth factor receptor 1 Antagonists may be useful in the promotion of angiogenesis. (isoform 1) (SEQ ID NO: 230) VEGF R-3; flt-4 GeneSeq Receptor for VEGF polypeptides VEGF activity, in the presence of flt-4 polypeptides, Accession B29047 can be determined using assays known in the art, such as those disclosed in WO0058511 International Publication No. WO0045835, for example. VEGF Receptor. Fusion P35916/VGFR3_HUMAN protein with the extracellular domain is useful as an anti-angiogenic agent. Vascular endothelial Antagonists may be useful in the promotion of angiogenesis. growth factor receptor 3 (isoform 1) (SEQ ID NO: 231) Neuropilin-1 GeneSeq Vascular Endothelial Growth Factor VEGF activity can be determined using assays Accession Y06319 known in the art, such as those disclosed in International Publication No. WO9929858 WO0045835, for example. Promotion of growth and proliferation of cells, such as O14786/NRP1_HUMAN vascular endothelial cells. Antagonists may be useful as anti-angiogenic agents, and Neuropilin-1 may be applicable for cancer. (isoform 1) (SEQ ID NO: 232) Neuropilin-2 GeneSeq Vascular Endothelial Growth Factor VEGF activity can be determined using assays Accession Y03618 known in the art, such as those disclosed in International Publication No. WO9929858 WO0045835, for example. Promotion of growth and proliferation of cells, such as O60462/NRP2_HUMAN vascular endothelial cells. Antagonists may be useful as anti-angiogenic agents, and Neuropilin-2 may be applicable for cancer. (isoform A22) (SEQ ID NO: 233) Human fast twitch skeletal Troponins are contractile proteins that are thought to inhibit angiogenesis. High levels muscle troponin C may contribute to the difficulty encountered in revascularizing the ischemic GeneSeq Accession myocardium after cardiovascular injury. Ability of soluble Troponins to inhibit W22597 W09730085 angiogenesis can be determined using assays known in the art:. Proc Natl Acad Sci P02585/TNNC2_HUMAN USA 1999 Mar. 16; 96(6): 2645-50. Anti-angiogenesis Troponin C, skeletal muscle (SEQ ID NO: 234) Human fast twitch skeletal Troponins are contractile proteins that are thought to inhibit angiogenesis. High levels muscle troponin I may contribute to the difficulty encountered in revascularizing the ischemic GeneSeq Accession myocardium after cardiovascular injury. Ability of soluble Troponins to inhibit W18054 W09730085 angiogenesis can be determined using assays known in the art. Proc Natl Acad Sci P48788/TNNI2_HUMAN USA 1999 Mar. 16; 96(6): 2645-50. Anti-angiogenesis Troponin I, fast skeletal muscle (isoform 1) (SEQ ID NO: 235) Human fast twitch skeletal Troponins are contractile proteins that are thought to inhibit angiogenesis. High levels muscle troponin T may contribute to the difficulty encountered in revascularizing the ischemic GeneSeq Accession myocardium after cardiovascular injury. Ability of soluble Troponins to inhibit W22599 W09730085 angiogenesis can be determined using assays known in the art:. Proc Natl Acad Sci SEQ ID NO: 3 of USA 1999 Mar. 16; 96(6): 2645-50. Anti-angiogenesis WO9730085 (SEQ ID NO: 236) fragment. myofibrillar Troponins are contractile proteins that are thought to inhibit angiogenesis. High levels protein troponin I may contribute to the difficulty encountered in revascularizing the ischemic GeneSeq Accession myocardium after cardiovascular injury. Ability of soluble Troponins to inhibit W18053 W09719955 angiogenesis can be determined using assays known in the art:. Proc Natl Acad Sci SEQ ID NO: 3 of USA 1999 Mar. 16; 96(6): 2645-50. Anti-angiogenesis WO9719955 (SEQ ID NO: 237) myofibrillar protein Troponins are contractile proteins that are thought to inhibit angiogenesis. High levels troponin I GeneSeq may contribute to the difficulty encountered in revascularizing the ischemic Accession W18054 myocardium after cardiovascular injury. Ability of soluble Troponins to inhibit WO9719955 angiogenesis can be determined using assays known in the art:. Proc Natl Acad Sci SEQ ID NO: 3 of USA 1999 Mar. 16; 96(6): 2645-50. Anti-angiogenesis WO9719955 (SEQ ID NO: 237) Troponin peptides Troponins are contractile proteins that are thought to inhibit angiogenesis. High levels GeneSeq Accessions may contribute to the difficulty encountered in revascularizing the ischemic Y29581, Y29582, Y29583, myocardium after cardiovascular injury. Ability of soluble Troponins to inhibit Y29584, Y29585, and angiogenesis can be determined using assays known in the art:. Proc Natl Acad Sci Y29586 WO9933874 USA 1999 Mar. 16; 96(6): 2645-50. Anti-angiogenesis Wildtype troponins provided as: Human fast twitch skeletal muscle troponin C GeneSeq Accession W22597 W09730085 P02585/TNNC2_HUMAN Troponin C, skeletal muscle (SEQ ID NO: 234) Human fast twitch skeletal muscle troponin I GeneSeq Accession W18054 W09730085 P48788/TNNI2_HUMAN Troponin I, fast skeletal muscle (isoform 1) (SEQ ID NO: 235) Human fast twitch skeletal muscle troponin T GeneSeq Accession W22599 W09730085 SEQ ID NO: 3 of WO9730085 (SEQ ID NO: 236) fragment. myofibrillar protein troponin I GeneSeq Accession W18053 W09719955 SEQ ID NO: 3 of WO9719955 (SEQ ID NO: 237) Human fast twitch skeletal muscle Troponin subunit C GeneSeq Accession B00134 WO0054770 SEQ ID NO: 1 of WO0054770 (SEQ ID NO: 375) Human fast twitch skeletal muscle Troponin subunit I Protein GeneSeq Accession B00135 WO0054770 SEQ ID NO: 2 of WO0054770 (SEQ ID NO: 376) Human fast twitch skeletal muscle Troponin subunit T GeneSeq Accession B00136 WO0054770 SEQ ID NO: 3 of WO0054770 (SEQ ID NO: 377) Human fast twitch skeletal Troponins are contractile proteins that are thought to inhibit angiogenesis. High levels muscle Troponin subunit C may contribute to the difficulty encountered in revascularizing the ischemic GeneSeq Accession myocardium after cardiovascular injury. Ability of soluble Troponins to inhibit B00134 WO0054770 angiogenesis can be determined using assays known in the art:. Proc Natl Acad Sci SEQ ID NO: 1 of USA 1999 Mar. 16; 96(6): 2645-50. Anti-angiogenesis WO0054770 (SEQ ID NO: 375) Human fast twitch skeletal Troponins are contractile proteins that are thought to inhibit angiogenesis. High levels muscle Troponin subunit I may contribute to the difficulty encountered in revascularizing the ischemic Protein GeneSeq myocardium after cardiovascular injury. Ability of soluble Troponins to inhibit Accession B00135 angiogenesis can be determined using assays known in the art:. Proc Natl Acad Sci WO0054770 USA 1999 Mar. 16; 96(6): 2645-50. Anti-angiogenesis SEQ ID NO: 2 of WO0054770 (SEQ ID NO: 376) Human fast twitch skeletal Troponins are contractile proteins that are thought to inhibit angiogenesis. High levels muscle Troponin subunit T may contribute to the difficulty encountered in revascularizing the ischemic GeneSeq Accession myocardium after cardiovascular injury. Ability of soluble Troponins to inhibit B00136 WO0054770 angiogenesis can be determined using assays known in the art:. Proc Natl Acad Sci SEQ ID NO: 3 of USA 1999 Mar. 16; 96(6): 2645-50. Anti-angiogenesis WO0054770 (SEQ ID NO: 377) Activator Inbibitor-1; PAI-1 PAIs are believed to play a role in cancer, and cardiovascular disease and blood- GeneSeq Accession clotting disorders. Methods that measure plasminogen activator inhibitor (PAI) activity R08411 WO9013648 are known in the art, for example, assay the ability of PAI to inhibit tissue P05121/PAI1_HUMAN plasminogen activator (tPA) or urokinase (uPA): J Biochem Biophys Methods 2000 Plasminogen activator Sep. 11; 45(2): 127-40, Breast Cancer Res Treat 1996; 41(2): 141-6. Methods that inhibitor 1 measure anti- angiogenesis activity are known in the art, for example, Proc Natl Acad (isoform 1) Sci USA 1999 Mar. 16; 96(6): 2645-50. Anti-angiogenesis; blood-clotting disorders. (SEQ ID NO: 238) Plasminogen Activator PAIs are believed to play a role in cancer, and cardiovascular disease and blood- Inhibitor-2; PAI-2 clotting disorders. Methods that measure plasminogen activator inhibitor (PAI) activity GeneSeq Accession are known in the art, for example, assay the ability of PAI to inhibit tissue P94160 DE3722673 plasminogen activator (tPA) or urokinase (uPA): J Biochem Biophys Methods 2000 P05120/PAI2_HUMAN Sep. 11; 45(2): 127-40, Breast Cancer Res Treat 1996; 41(2): 141-6. Methods that Plasminogen activator measure anti- angiogenesis activity are known in the art, for example, Proc Natl Acad inhibitor 2 Sci USA 1999 Mar. 16; 96(6): 2645-50. Anti-angiogenesis; blood-clotting disorders. (SEQ ID NO: 239) Activator Inhibitor-2; PAI-2 PAIs are believed to play a role in cancer, and cardiovascular disease and blood- GeneSeq Accession clotting disorders. Methods that measure plasminogen activator inhibitor (PAI) activity R10921 WO9102057 are known in the art, for example, assay the ability of PAI to inhibit tissue P05120/PAI2_HUMAN plasminogen activator (tPA) or urokinase (uPA): J Biochem Biophys Methods 2000 Plasminogen activator Sep. 11; 45(2): 127-40, Breast Cancer Res Treat 1996; 41(2): 141-6. Methods that inhibitor 2 measure anti- angiogenesis activity are known in the art, for example, Proc Natl Acad (SEQ ID NO: 239) Sci USA 1999 Mar. 16; 96(6): 2645-50. Anti-angiogenesis; blood-clotting disorders. Human PAI-1 mutants PAIs are believed to play a role in cancer, and cardiovascular disease and blood- GeneSeq Accessions clotting disorders. Methods that measure plasminogen activator inhibitor (PAI) activity R11755, R11756, R11757, are known in the art, for example, assay the ability of PAI to inhibit tissue R11758, R11759, R11760, plasminogen activator (tPA) or urokinase (uPA): J Biochem Biophys Methods 2000 R11761, R11762 and Sep. 11; 45(2): 127-40, Breast Cancer Res Treat 1996; 41(2): 141-6. Methods that R11763 WO9105048 measure anti- angiogenesis activity are known in the art, for example, Proc Natl Acad Wildtype PAI-1 is provided Sci USA 1999 Mar. 16; 96(6): 2645-50. Anti-angiogenesis; blood-clotting disorders. as P05121/PAI1_HUMAN Plasminogen activator inhibitor 1 (isoform 1) (SEQ ID NO: 238) CXCR3; CXC GeneSeq Chemokines are a family of related small, secreted proteins involved in biological Accession Y79372 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. WO0018431 Members of this family are involved in a similarly diverse range of pathologies P49682|CXCR3_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The C-X-C chemokine receptor chemokines exert their effects by acting on a family of seven transmembrane G- type 3 protein coupled receptors. Over 40 human chemokines have been described, which (isoform 1) bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 240) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Soluble CXCR3 polypeptides may be useful for inhibiting chemokine activities and viral infection. Modified Rantes GeneSeq Chemokines are a family of related small, secreted proteins involved in biological Accession W38129 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. WO9737005 Members of this family are involved in a similarly diverse range of pathologies Wildtype Rantes provided including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The herein as chemokines exert their effects by acting on a family of seven transmembrane G- P13501/CCL5_HUMAN protein coupled receptors. Over 40 human chemokines have been described, which C-C motif chemokine 5 bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 241) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders. RANTES GeneSeq Chemokines are a family of related small, secreted proteins involved in biological Accession Y05299 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. EP905240 Members of this family are involved in a similarly diverse range of pathologies P13501/CCL5_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The C-C motif chemokine 5 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 241) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders. MCP-Ia GeneSeq Chemokines are a family of related small, secreted proteins involved in biological Accession R73914 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. WO9509232 Members of this family are involved in a similarly diverse range of pathologies MCP-1 provided as including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The P13500/CCL2_HUMAN chemokines exert their effects by acting on a family of seven transmembrane G- C-C motif chemokine 2 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 337) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders. MCP-Ib GeneSeq Chemokines are a family of related small, secreted proteins involved in biological Accession Y26176 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. WO9929728 Members of this family are involved in a similarly diverse range of pathologies MCP-1 provided as including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The P13500/CCL2_HUMAN chemokines exert their effects by acting on a family of seven transmembrane G- C-C motif chemokine 2 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 337) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders. MCP-I receptor GeneSeq Chemokines are a family of related small, secreted proteins involved in biological Accession R79165 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. WO9519436 Members of this family are involved in a similarly diverse range of pathologies MCP-IA including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The SEQ ID NO: 2 of chemokines exert their effects by acting on a family of seven transmembrane G- WO9519436 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 446) bind to ~17 receptors thus far identified. Chemokine activities can be determined MCP-1B using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: SEQ ID NO: 4 of Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. WO9519436 Humana Press Inc., Totowa, NJ. Soluble MCP-1 Receptor polypep- tides may be (SEQ ID NO: 447) useful for inhibiting chemokine activities and viral infection. MCP-3 GeneSeq Chemokines are a family of related small, secreted proteins involved in biological Accession R73915 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. W09509232 Members of this family are involved in a similarly diverse range of pathologies P80098/CCL7_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The C-C motif chemokine 7 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 336) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders. MCP-4 receptor GeneSeq Chemokines are a family of related small, secreted proteins involved in biological Accession W56689 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. WO9809171 Members of this family are involved in a similarly diverse range of pathologies SEQ ID NO: 2 of including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The WO9809171 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 378) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Soluble MCP-4 Receptor polypep- tides may be useful for inhibiting chemokine activities and viral infection. RANTES receptor Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. W29588 U.S. Pat. No. Members of this family are involved in a similarly diverse range of pathologies 5,652,133 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The SEQ ID NO: 2 of U.S. Pat. chemokines exert their effects by acting on a family of seven transmembrane G- No. 5,652,133 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 379) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Soluble RANTES Receptor polypep- tides may be useful for inhibiting chemokine activities and viral infection. CCR5 variant GeneSeq Chemokines are a family of related small, secreted proteins involved in biological Accession W88238 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. WO9854317 Members of this family are involved in a similarly diverse range of pathologies Variants of wildtype CCR5 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The which has the sequence chemokines exert their effects by acting on a family of seven transmembrane G- of: protein coupled receptors. Over 40 human chemokines have been described, which P51681|CCR5_HUMAN bind to ~17 receptors thus far identified. Chemokine activities can be determined C-C chemokine receptor using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: type 5 Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. (SEQ ID NO: 448) Humana Press Inc., Totowa, NJ. Soluble CCR5 polypeptides may be useful for inhibiting chemokine activities and viral infection. CCR7 GeneSeq Chemokines are a family of related small, secreted proteins involved in biological Accession B50859 U.S. processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Pat. No. 6,153,441 Members of this family are involved in a similarly diverse range of pathologies P32248/CCR7_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The C-C chemokine receptor chemokines exert their effects by acting on a family of seven transmembrane G- type 7 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 243) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Soluble CCR7 polypeptides may be useful for inhibiting chemokine activities and viral infection. CXC3 GeneSeq Chemokines are a family of related small, secreted proteins involved in biological Accession W23345 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. WO9727299 Members of this family are involved in a similarly diverse range of pathologies P78423/X3CL1_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The Fractalkine chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 244) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders. Eotaxin GeneSeq Chemokines are a family of related small, secreted proteins involved in biological Accession W10099 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. WO9700960 Members of this family are involved in a similarly diverse range of pathologies P51671/CCL11_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The Eotaxin chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 245) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders. Neurotactin GeneSeq Neurotactin may play a role in chemotactic leukocyte migration and brain Accessions Y77537, inflammation processes. Chemotactic leukocyte migration assays are known in the W34307, Y53259, and, art, for example: J. Immunol. Methods 33, ((1980)); Nature 1997 Jun. 5; 387(6633): Y77539 U.S. Pat. No. 611-7. Immune disorders. 6,013,257 WO9742224 P78423/X3CL1_HUMAN Fractalkine (SEQ ID NO: 244) Human CKbeta-9 Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. B50860 U.S. Pat. No. Members of this family are involved in a similarly diverse range of pathologies 6,153,441 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The SEQ ID NO: 2 of U.S. Pat. chemokines exert their effects by acting on a family of seven transmembrane G- No. 6,153,441 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 246) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders. Lymphotactin GeneSeq Chemokines are a family of related small, secreted proteins involved in biological Accession B50052 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. WO0073320 Members of this family are involved in a similarly diverse range of pathologies P47992/XCL1_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The Lymphotactin chemokines exert their effects by acting on a family of seven transmembrane G. (SEQ ID NO: 247) Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders. MIP-3 alpha GeneSeq Chemokines are a family of related small, secreted proteins involved in biological Accession W44398 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. WO9801557 Members of this family are involved in a similarly diverse range of pathologies P78556/CCL20_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The C-C motif chemokine 20 chemokines exert their effects by acting on a family of seven transmembrane G. (isoform 1) Chemokine activities can be determined using assays known in the art: Methods in (SEQ ID NO: 248) Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders. MIP-3 beta GeneSeq Chemokines are a family of related small, secreted proteins involved in biological Accession W44399 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. WO9801557 Members of this family are involved in a similarly diverse range of pathologies Q99731/CCL19_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The C-C motif chemokine 19 chemokines exert their effects by acting on a family of seven transmembrane G. (SEQ ID NO: 249) Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders. MIP-Gamma GeneSeq Chemokines are a family of related small, secreted proteins involved in biological Accession R70798 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. WO2006135382 Members of this family are involved in a similarly diverse range of pathologies (SEQ ID NO: 457) including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The chemokines exert their effects by acting on a family of seven transmembrane G. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders. Stem Cell Inhibitory Factor Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. R11553 WO9104274 Members of this family are involved in a similarly diverse range of pathologies SCIF in Table I of including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The WO9104274 chemokines exert their effects by acting on a family of seven transmembrane G. (SEQ ID NO: 380) Chemokine activities can be determined using assays known in the art: Methods in SCIF in Table II of Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, WO9104274 T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. (SEQ ID NO: 381) Hematopoietic growth factors. Thrombopoietin GeneSeq Thrombopoietin is involved in the regulation of the growth and differentiation of Accession R79905 megakaryocytes and preceptors thereof. Thrombopoietin (TPO) can be assayed to WO9521920 determine regulation of growth and differentiation of megakaryocytes. Mol Cell Biol P40225|TPO_HUMAN 2001 April; 21(8): 2659-70; Exp Hematol 2001 January; 29(1): 51-8 and within. Thrombopoietin Hematopoietic growth factors. (isoform 1) (SEQ ID NO: 250) c-kit ligand; SCF; Mast cell C-kit ligan is thought to stimulate the proliferation of mast cells, and is able to growth factor; MGF; augment the proliferation of both myeloid and lymphoid hematopoietic progenitors in Fibrosarcoma- derived bone marrow culture. C-kit ligand is also though to act synergistically with other stem cell factor GeneSeq cytokines. Chemokine activities can be determined using assays known in the art: Accession Y53284, Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, R83978 and R83977 T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. EP992579 and EP676470 Hematopoietic growth factors. P21583|SCF_HUMAN Kit ligand (isoform 1) (SEQ ID NO: 251) Platelet derived growth Vascular Endothelial Growth Factor VEGF activity can be determined using assays factor GeneSeq Accession known in the art, such as those disclosed in International Publication No. B48653 WO0066736 WO0045835, for example. Promotion of growth and proliferation of cells, such as PDGF-A vascular endothelial cells. Antagonists may be useful as anti-angiogenic agents, and P04085/PDGFA_HUMAN may be applicable for cancer. Platelet-derived growth factor subunit A (Isoform long) (SEQ ID NO: 257) PDGF-B P01127/PDGFB_HUMAN Platelet-derived growth factor subunit B (isoform 1) (SEQ ID NO: 258) Melanoma inhibiting Melanoma inhibiting protein has melanoma-inhibiting activity and can be used to treat protein GeneSeq cancer (melanoma, glioblastoma, neuroblastoma, small cell lung cancer, Accession R69811 neuroectodermal tumors) or as an immunosuppressant (it inhibits IL-2 or WO9503328 phytohaemagglutinin induced proliferation of peripheral blood lymphocytes. Tumor (SEQ ID NO: 458) suppressor activity of melanoma inhibiting protein can be determined using assays known in the art: Matzuk et al., Nature 1992 Nov. 26; 360(6402): 313-9. Cancer; melanoma Glioma-derived growth Vascular Endothelial Growth Factor VEGF activity can be determined using assays factor GeneSeq Accession known in the art, such as those disclosed in International Publication No. R08120 EP399816 WO0045835, for example. Promotion of growth and proliferation of cells, such as vascular endothelial cells. Antagonists may be useful as anti-angiogenic agents, and may be applicable for cancer. Platelet derived growth Vascular Endothelial Growth Factor VEGF activity can be determined using assays factor precursor A known in the art, such as those disclosed in International Publication No. GeneSeq Accession WO0045835, for example. Promotion of growth and proliferation of cells, such as R84759 EP682110 vascular endothelial cells. Antagonists may be useful as anti-angiogenic agents, and PDGF-A precursor (variant may be applicable for cancer. D1) (SEQ ID NO: 382) PDGF-A precursor (variant 13-1) (SEQ ID NO: 383) Platelet derived growth Vascular Endothelial Growth Factor VEGF activity can be determined using assays factor precursor B known in the art, such as those disclosed in International Publication No. GeneSeq Accession WO0045835, for example. Promotion of growth and proliferation of cells, such as R84760 EP682110, FIG. vascular endothelial cells. Antagonists may be useful as anti-angiogenic agents, and 1 or FIG. 2 may be applicable for cancer. Wildtype PDGF-B provided as: PDGF-B P01127/PDGFB_HUMAN Platelet-derived growth factor subunit B (isoform 1) (SEQ ID NO: 258) Platelet derived growth Vascular Endothelial Growth Factor VEGF activity can be determined using assays factor Bvsis GeneSeq known in the art, such as those disclosed in International Publication No. Accession P80595 and WO0045835, for example. Promotion of growth and proliferation of cells, such as P80596 EP282317 vascular endothelial cells. Antagonists may be useful as anti-angiogenic agents, and FIG. 1 of EP282317 may be applicable for cancer. (SEQ ID NO: 384) Placental Growth Factor Vascular Endothelial Growth Factor VEGF activity can be determined using assays GeneSeq Accessions known in the art, such as those disclosed in International Publication No. R23059 and R23060 WO0045835, for example. Promotion of growth and proliferation of cells, such as WO9206194 vascular endothelial cells. Antagonists may be useful as anti-angiogenic agents, and P49763-2/PLGF_HUMAN may be applicable for cancer. Isoform PIGF-1 of Placenta growth factor (isoform PIGF-1) (SEQ ID NO: 252) Placental Growth Factor-2 Vascular Endothelial Growth Factor VEGF activity can be determined using assays GeneSeq Accession known in the art, such as those disclosed in International Publication No. Y08289 DE19748734 WO0045835, for example. Promotion of growth and proliferation of cells, such as P49763-3/PLGF_HUMAN vascular endothelial cells. Antagonists may be useful as anti-angiogenic agents, and Isoform PIGF-2 of may be applicable for cancer. Placenta growth factor (isoform PIGF-2) (SEQ ID NO: 253) Thrombopoietin Thrombopoietin is involved in the regulation of the growth and differentiation of derivative1 GeneSeq megakaryocytes and preceptors thereof. Thrombopoietin (TPO) can be assayed to Accession Y77244 determine regulation of growth and differentiation of megakaryocytes. Mol Cell Biol WO0000612 (e.g. Table 3) 2001 April; 21(8): 2659-70; Exp Hematol 2001 January; 29(1): 51-8 and within. Wildtype thrombopoietin Thrombocytopenia, cancer. provided as: P40225|TPO_HUMAN Thrombopoietin (isoform 1) (SEQ ID NO: 250) Thrombopoietin Thrombopoietin is involved in the regulation of the growth and differentiation of derivative2 GeneSeq megakaryocytes and preceptors thereof. Thrombopoietin (TPO) can be assayed to Accession Y77255 determine regulation of growth and differentiation of megakaryocytes. Mol Cell Biol WO0000612 (e.g. Table 3) 2001 April; 21(8): 2659-70; Exp Hematol 2001 January; 29(1): 51-8 and within. Wildtype thrombopoietin Thrombocytopenia, cancer. provided as: P40225|TPO_HUMAN Thrombopoietin (isoform 1) (SEQ ID NO: 250) Thrombopoietin derivative Thrombopoietin is involved in the regulation of the growth and differentiation of 3 GeneSeq Accession megakaryocytes and preceptors thereof. Thrombopoietin (TPO) can be assayed to Y77262 determine regulation of growth and differentiation of megakaryocytes. Mol Cell Biol WO0000612 (e.g. Table 3) 2001 April; 21(8): 2659-70; Exp Hematol 2001 January; 29(1): 51-8 and within. Wildtype thrombopoietin Thrombocytopenia, cancer. provided as: P40225|TPO_HUMAN Thrombopoietin (isoform 1) (SEQ ID NO: 250) Thrombopoietin derivative Thrombopoietin is involved in the regulation of the growth and differentiation of 4 GeneSeq Accession megakaryocytes and preceptors thereof. Thrombopoietin (TPO) can be assayed to Y77267 determine regulation of growth and differentiation of megakaryocytes. Mol Cell Biol WO0000612 (e.g. Table 3) 2001 April; 21(8): 2659-70; Exp Hematol 2001 January; 29(1): 51-8 and within. Wildtype thrombopoietin Thrombocytopenia, cancer. provided as: P40225|TPO_HUMAN Thrombopoietin (isoform 1) (SEQ ID NO: 250) Thrombopoietin derivative Thrombopoietin is involved in the regulation of the growth and differentiation of 5 GeneSeq Accession megakaryocytes and preceptors thereof. Thrombopoietin (TPO) can be assayed to Y77246 determine regulation of growth and differentiation of megakaryocytes. Mol Cell Biol WO0000612 (e.g. Table 3) 2001 April; 21(8): 2659-70; Exp Hematol 2001 January; 29(1): 51-8 and within. Wildtype thrombopoietin Thrombocytopenia, cancer. provided as: P40225|TPO_HUMAN Thrombopoietin (isoform 1) (SEQ ID NO: 250) Thrombopoietin derivative Thrombopoietin is involved in the regulation of the growth and differentiation of 6 GeneSeq Accession megakaryocytes and preceptors thereof. Thrombopoietin (TPO) can be assayed to Y77253 determine regulation of growth and differentiation of megakaryocytes. Mol Cell Biol WO0000612 (e.g. Table 3) 2001 April; 21(8): 2659-70; Exp Hematol 2001 January; 29(1): 51-8 and within. Wildtype thrombopoietin Thrombocytopenia, cancer. provided as: P40225|TPO_HUMAN Thrombopoietin (isoform 1) (SEQ ID NO: 250) Thrombopoietin derivative Thrombopoietin is involved in the regulation of the growth and differentiation of 7 GeneSeq Accession megakaryocytes and preceptors thereof. Thrombopoietin (TPO) can be assayed to Y77256 determine regulation of growth and differentiation of megakaryocytes. Mol Cell Biol WO0000612 (e.g. Table 3) 2001 April; 21(8): 2659-70; Exp Hematol 2001 January; 29(1): 51-8 and within. Wildtype thrombopoietin Thrombocytopenia, cancer. provided as: P40225|TPO_HUMAN Thrombopoietin (isoform 1) (SEQ ID NO: 250) Fractalkine GeneSeq Fractalkine is believed to play a role in chemotactic leukocyte migration and Accession Y53255 U.S. neurological disorders. Fractalkine activity can be determined using Chemotactic Pat. No. 6,043,086 leukocyte migration assays known in the art, for example: J. Immunol. Methods 33, P78423/X3CL1_HUMAN ((1980)); Nature 1997 Jun. 5; 387(6633): 611-7. Immune disorders. Fractalkine (SEQ ID NO: 244) CXC3 GeneSeq Chemokines are a family of related small, secreted proteins involved in biological Accession W23345 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. WO9757599 Members of this family are involved in a similarly diverse range of pathologies P78423/X3CL1_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The Fractalkine chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 244) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders. CCR7 GeneSeq Chemokines are a family of related small, secreted proteins involved in biological Accession B50859 U.S. processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Pat. No. 6,153,441 Members of this family are involved in a similarly diverse range of pathologies P32248/CCR7_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The C-C chemokine receptor chemokines exert their effects by acting on a family of seven transmembrane G- type 7 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 243) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Soluble CCR7 polypeptides may be useful for inhibiting chemokine activities and viral infection. Nerve Growth Factor-beta Nerve Growth Factor Proliferation assay using NR6R-3T3 cells (Rizzino 1988 Cancer GeneSeq Accession Res. 48: 4266) Neurological disorders, cancer R11474 EP414151 P01138/NGF_HUMAN Beta-nerve growth factor (SEQ ID NO: 254) Nerve Growth Factor- Nerve Growth Factor Proliferation assay using NR6R 3T3 cells (Rizzino 1988 Cancer beta2 GeneSeq Accession Res. 48: 4266 Neurological disorders, cancer W69725 EP859056 FIG. 1 of EP859056 (SEQ ID NO: 465) Neurotrophin-3 GeneSeq Neurotrophins regulate neuronal cell survival and synaptic plasticity. Trk tyrosine Accession W8889 kinase activation assays known in the art can be used to assay for neurotrophin WO9821234 activity, for example, Proc Natl Acad Sci USA 2001 Mar. 13; 98(6): 3555-3560. P20783/NTF3_HUMAN Neurological disorders, cancer Neurotrophin-3 (isoform 1) (SEQ ID NO: 255) Neurotrophin-4 GeneSeq Neurotrophins regulate neuronal cell survival and synaptic plasticity. Trk tyrosine Accession R47100 kinase activation assays known in the art can be used to assay for neurotrophin WO9325684 activity, for example, Proc Natl Acad Sci USA 2001 Mar. 13; 98(6): 3555-3560. P34130/NTF4_HUMAN Neurological disorders, cancer Neurotrophin-4 (SEQ ID NO: 256) Neurotrophin- 4a Neurotrophins regulate neuronal cell survival and synaptic plasticity. Trk tyrosine GeneSeq Accession kinase activation assays known in the art can be used to assay for neurotrophin R47101 WO9325684 activity, for example, Proc Natl Acad Sci USA 2001 Mar. 13; 98(6): 3555-3560. Wildtype neurotrophin Neurological disorders, cancer provided as: P34130/NTF4_HUMAN Neurotrophin-4 (SEQ ID NO: 256) Neurotrophin- 4b Neurotrophins regulate neuronal cell survival and synaptic plasticity. tyrosine kinases. GeneSeq Accession Trk tyrosine kinase activation assays known in the art can be used to assay for R47102 WO9325684 neurotrophin activity, for example, Proc Natl Acad Sci USA 2001 Mar. 13; 98(6): 3555-3560. P34130/NTF4_HUMAN Neurological disorders, cancer Neurotrophin-4 (SEQ ID NO: 256) Neurotrophin- 4c Neurotrophins regulate neuronal cell survival and synaptic plasticity. tyrosine kinases. GeneSeq Accession Trk tyrosine kinase activation assays known in the art can be used to assay for R47103 WO9325684 neurotrophin activity, for example, Proc Natl Acad Sci USA 2001 Mar. 13; 98(6): 3555-3560. P34130/NTF4_HUMAN Neurological disorders, cancer Neurotrophin-4 (SEQ ID NO: 256) Neurotrophin- 4d Neurotrophins regulate neuronal cell survival and synaptic plasticity. tyrosine kinases. GeneSeq Accession Trk tyrosine kinase activation assays known in the art can be used to assay for R47102 WO9325684 neurotrophin activity, for example, Proc Natl Acad Sci USA 2001 Mar. 13; 98(6): 3555-3560. P34130/NTF4_HUMAN Neurological disorders, cancer Neurotrophin-4 (SEQ ID NO: 256) Platelet-Derived Growth Vascular Endothelial Growth Factor VEGF activity can be determined using assays Factor A chain GeneSeq known in the art, such as those disclosed in International Publication No. Accession R38918 U.S. W00045835, for example. Promotion of growth and proliferation of cells, such as Pat. No. 5,219,739 vascular endothelial cells. Hematopoietic and immune dis- orders. Antagonists may P04085/PDGFA_HUMAN be useful as anti-angiogenic agents, and may be applicable for cancer Platelet-derived growth factor subunit A (Isoform long) (SEQ ID NO: 257) Platelet-Derived Growth Vascular Endothelial Growth Factor VEGF activity can be determined using assays Factor B chain GeneSeq known in the art, such as those disclosed in International Publication No. Accession R38919 U.S. W00045835, for example. Promotion of growth and proliferation of cells, such as Pat. No. 5,219,739 vascular endothelial cells. Hematopoietic and immune dis- orders. Antagonists may P01127/PDGFB_HUMAN be useful as anti-angiogenic agents, and may be applicable for cancer Platelet-derived growth factor subunit B (isoform 1) (SEQ ID NO: 258) Stromal Derived Factor- 1 Stromal Growth Factor Proliferation assay using NR6R-3T3 cells (Rizzino 1988 alpha GeneSeq Accession Cancer Res. 48: 4266) Hematopoietic, immune disorders, cancer Y39995 WO9948528 P48061-2/SDF1_HUMAN Isoform Alpha of Stromal cell-derived factor 1 (isoform alpha) (SEQ ID NO: 259) Stromal Derived Factor- 1 Stromal Growth Factor Proliferation assay using NR6R-3T3 cells (Rizzino 1988 beta GeneSeq Accession Cancer Res. 48: 4266) Hematopoietic, immune disorders, cancer R75420 CA2117953 P48061/SDF1_HUMAN Stromal cell-derived factor 1 (isoform beta) (SEQ ID NO: 260) Tarc GeneSeq Accession Chemotactic for T lymphocytes. May play a role in T-cell development. Thought to W14917 WO9711969 bind CCR8 and CCR4 Chemotactic leukocyte migration assays are known in the art, Q92583/CCL17_HUMAN for example: J. Immunol. Methods 33 ((1980)) Antiinflammatory. Immune disorders, C-C motif chemokine 17 cancer (SEQ ID NO: 261) Prolactin GeneSeq Prolactin is involved in immune cell proliferation and apoptosis. Immune coil Accession R78691 proliferation and suppression of apoptosis by prolactin can be assayed by methods WO9521625 well-known in the art, for example, Buckley, A R and Buckley D J, Ann NY Acad Sci P01236/PRL_HUMAN 2000; 917: 522-33, and within. Reproductive system disorders, cancer. Prolactin (SEQ ID NO: 262) Prolactin2 GeneSeq Prolactin is involved in immune cell proliferation and apoptosis. Immune coil Accession Y31764 U.S. proliferation and suppression of apoptosis by prolactin can be assayed by methods Pat. No. 5,955,346 well-known in the art, for example, Buckley, A R and Buckley D J, Ann NY Acad Sci 2000; 917: 522-33, and within. Reproductive system disorders, cancer. Follicle stimulating FSH stimulates secretion of interleukin-1 by cells isolated from women in the follicular hormone Alpha subunit phase FSH activities can be determined using assays known in the art; J Gend Specif GeneSeq Accession Med 1999 November-December; 2(6): 30-4; Mol Cell Endocrinol. 1997 Nov. 15; 134(2): 109-18. Y54160 EP974359 Reproductive system disorders, cancer. P01215/GLHA_HUMAN Glycoprotein hormones alpha chain (SEQ ID NO: 263) Follicle stimulating FSH stimulates secretion of interleukin-1 by cells isolated from women in the follicular hormone Beta subunit phase FSH activities can be determined using assays known in the art; J Gend Specif GeneSeq Accession Med 1999 November-December; 2(6): 30-4; Mol Cell Endocrinol. 1997 Nov. 15; 134(2): 109-18. Y54161 EP974359 Reproductive system disorders, cancer. P01225/FSHB_HUMAN Follitropin subunit beta (SEQ ID NO: 264) Substance P (tachykinin) Substance P is associated with immunoregulation. Immuneregulation and bone GeneSeq Accession marrow, cell proliferation by substance P can be assayed by methods well-known in B23027 WO0054053 the art, for example, Lai et al. Proc Natl Acad Sci USA 2001 Mar. 27; 98(7): 3970-5; (SEQ ID NO: 385) Jallat-Daloz et al. Allergy Asthma Proc 2001 January-February; 22(1): 17-23; Kehler et al. Exp Lung Res 2001 January-February; 27(1): 25-46; and Adamus M A and Dabrowski Z J. J Cell Biochem 2001; 81(3)499-506. diabetes mellitus, hypertension, cancer Oxytocin (Neurophysin I) Oxytocin is involved in the induction of prostaglandin (E2) release as well as an GeneSeq Accession increased amount of calcium release by smooth muscle cells. Oxytocin and B24085 and B24086 prostaglandin E(2) release and Ocytocin (Ca2+) increase can be assayed by WO0053755 methods well-known in the art, for example, Pavan et al., AM J Obset Gynecol 2000 P01178/NEU1_HUMAN July; 183(1): 76-82 and Holda et al., Cell Calcium 1996 July; 20(1): 43 51. inflammatory Oxytocin-neurophysin 1 disorders immunologic disorders, cancer (SEQ ID NO: 265) Vasopressin (Neurophysin Vasopressinis believed to have a direct antidiuretic action on the kidney, and it is II) GeneSeq Accession thought to cause vasoconstriction of the peripheral vessels. Vasopressin activity can B24085 and B24086 be determined using assays known in the art, for example, Endocr Regul 1996 March; WO0053755 30(I): 13-17. inflammatory disorders immunologic disorders, cancer P01185/NEU2_HUMAN Vasopressin-neurophysin 2-copeptin (SEQ ID NO: 266) IL-1 GeneSeq Accession Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, P60326 EP165654 monocytes, and macrophages. Known functions include stimulating proliferation of IL-1 alpha immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis P01583|IL1A_HUMAN of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Interleukin-1 alpha can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 269) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, IL-1 beta D.C. 1987, pp. 221-225; and Orencole & Dinarclio (1989) Cytokine 1, 14-20. P01584|IL1B_HUMAN inflammatory disorders immunologic disorders, cancer Interleukin-1 beta (SEQ ID NO: 267) IL-1 mature GeneSeq Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, Accession R14855 monocytes, and macrophages. Known functions include stimulating proliferation of EP456332 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis (mature truncated form of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity wherein the precursor is can be determined using assays known in the art: Matthews et al., in Lymphokines cleaved between amino and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, acids 116-117) D.C. 1987, pp. 221-225; and Orencole & Dinarclio (1989) Cytokine 1, 14-20. (SEQ ID NO: 386) inflammatory disorders immunologic disorders, cancer IL-1 beta GeneSeq Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, Accession Y08322 monocytes, and macrophages. Known functions include stimulating proliferation of WO9922763 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis P01584|IL1B_HUMAN of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Interleukin-1 beta can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 267) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Orencole & Dinarclio (1989) Cytokine 1, 14-20. inflammatory disorders immunologic disorders, cancer IL-3 variants GeneSeq Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, Accession P80382, monocytes, and macrophages. Known functions include stimulating proliferation of P80383, P80384, and immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis P80381 WO8806161 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Variants of wildtype IL-3 can be determined using assays known in the art: Matthews et al., in Lymphokines which has the sequence: and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, P08700|IL3_HUMAN D.C. 1987, pp. 221-225; and Kitamura et al (1989) J Cell Physiol. 140 323-334. Interleukin-3 inflammatory disorders immunologic disorders, cancer (SEQ ID NO: 449) IL-4 GeneSeq Accession Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, P70615 WO8702990 monocytes, and macrophages. Known functions include stimulating proliferation of P05112/IL4_HUMAN immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis Interleukin-4 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity (isoform 1) can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 268) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Siegel & Mostowski (1990) J Immunol Methods 132, 287-295. inflammatory disorders immunologic disorders, cancer IL-4 muteins GeneSeq Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, Accession W52151 monocytes, and macrophages. Known functions include stimulating proliferation of W52152 W52153 W52154 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis W52155 W52156 W52157 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity W52158 W52159 W52160 can be determined using assays known in the art: Matthews et al., in Lymphokines W52161 W52162 W52163 and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, W52164 and W52165 D.C. 1987, pp. 221-225; and Siegel & Mostowski (1990) J Immunol Methods 132, WO9747744 287-295. inflammatory disorders immunologic disorders, cancer Variants of wildtype IL-4 which has the sequence: P05112/IL4_HUMAN Interleukin-4 (isoform 1) (SEQ ID NO: 268) IL-1 alpha GeneSeq Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, Accession P90108 monocytes, and macrophages. Known functions include stimulating proliferation of EP324447 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis P01583|IL1A_HUMAN of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Interleukin-1 alpha can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 269) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Orencole & Dinarclio (1989) Cytokine 1, 14-20. inflammatory disorders immunologic disorders, cancer IL-3 variants GeneSeq Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, Accession R38561, monocytes, and macrophages. Known functions include stimulating proliferation of R38562, R38563, R38564, immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis R38565, R38566, R38567, of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity R38568, R38569, R38570, can be determined using assays known in the art: Matthews et al., in Lymphokines R38571, and R38572 and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, WO9307171 D.C. 1987, pp. 221-225; and Aarden et al (1987) Eur. J. Immunol 17, 1411-16. Variants of wildtype IL-3 inflammatory disorders immunologic disorders, cancer which has the sequence: P08700|IL3_HUMAN Interleukin-3 (SEQ ID NO: 449) IL-6 GeneSeq Accession Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, R45717 and R45718 monocytes, and macrophages. Known functions include stimulating proliferation of WO9402512 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis P05231/IL6_HUMAN of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Interleukin-6 can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 270) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Aarden et al (1987) Eur. J. Immunol 17, 1411-16. inflammatory disorders immunologic disorders, cancer. Obesity. Metabolic Disease. Diabetes. IL-13 GeneSeq Accession Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, R48624 WO9404680 monocytes, and macrophages. Known functions include stimulating proliferation of P35225/IL13_HUMAN immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis Interleukin-13 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity (SEQ ID NO: 271) can be determined using assays known in the art: Matthews et al., in Lymphokines and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Boutelier et al (1995) J. Immunol. Methods 181, 29. inflammatory disorders immunologic disorders, cancer IL-4 mutein GeneSeq Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, Accession R47182 monocytes, and macrophages. Known functions include stimulating proliferation of DE4137333 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis Variants of wildtype IL-4 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity which has the sequence: can be determined using assays known in the art: Matthews et al., in Lymphokines P05112/IL4_HUMAN and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, Interleukin-4 D.C. 1987, pp. 221-225; and Siegel & Mostowski (1990) J Immunol Methods 132, (isoform 1) 287-295. inflammatory disorders immunologic disorders, cancer (SEQ ID NO: 268) IL-4 mutein Y124X Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of R47183 DE4137333 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis Variants of wildtype IL-4 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity which has the sequence: can be determined using assays known in the art: Matthews et al., in Lymphokines P05112/IL4_HUMAN and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, Interleukin-4 D.C. 1987, pp. 221-225; and Siegel & Mostowski (1990) J Immunol Methods 132, (isoform 1) 287-295. inflammatory disorders immunologic disorders, cancer (SEQ ID NO: 268)) IL-4 mutein Y124G Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of R47184 DE4137333 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis Variants of wildtype IL-4 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity which has the sequence: can be determined using assays known in the art: Matthews et al., in Lymphokines P05112/IL4_HUMAN and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, Interleukin-4 D.C. 1987, pp. 221-225; and Siegel & Mostowski (1990) J Immunol Methods 132, (isoform 1) 287-295. inflammatory disorders immunologic disorders, cancer (SEQ ID NO: 268) Human Interleukin-10 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, (precursor) GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession R41664 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis WO9317698 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity P22301/IL10_HUMAN can be determined using assays known in the art: Matthews et al., in Lymphokines Interleukin-10 and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (precursor form is D.C. 1987, pp. 221-225; and Thompson-Snipes et al (1991) J. Exp. Med. 173, 507-510. processed into a truncated inflammatory disorders immunologic disorders, cancer mature form) (SEQ ID NO: 272) Human Interleukin-10 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of R42642 WO9318783-A immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis SEQ ID NO: 3 of of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity WO9318783-A can be determined using assays known in the art: Matthews et al., in Lymphokines (mature IL-10) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (SEQ ID NO: 273) D.C. 1987, pp. 221-225; and Thompson-Snipes et al (1991) J. Exp. Med. 173, 507-510. inflammatory disorders immunologic disorders, cancer Human interleukin-1 beta Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, precursor. GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession R42447 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis EP569042 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity P01584/IL1B_HUMAN can be determined using assays known in the art: Matthews et al., in Lymphokines Interleukin-1 beta and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (SEQ ID NO: 274) D.C. 1987, pp. 221-225; and Orencole & Dinarclio (1989) Cytokine 1, 14-20. inflammatory disorders immunologic disorders, cancer Interleukin- 1alpha Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of R45364 EP578278 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis P01583|IL1A_HUMAN of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Interleukin-1 alpha can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 269) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225. inflammatory disorders immunologic disorders, cancer Human interleukin-3 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, variant GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession R22814 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis JP04063595 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Variants of wildtype IL-3 can be determined using assays known in the art: Matthews et al., in Lymphokines which has the sequence: and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, P08700|IL3_HUMAN D.C. 1987, pp. 221-225; Kitamura et al (1989) J Cell Physiol. 140 323-334. Interleukin-3 inflammatory disorders immunologic disorders, cancer (SEQ ID NO: 449) IL-1i fragments GeneSeq Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, Accession R35484 and monocytes, and macrophages. Known functions include stimulating proliferation of R35485 EP541920 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity can be determined using assays known in the art: Matthews et al., in Lymphokines and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Orencole & Dinarclio (1989) Cytokine 1, 14-20. inflammatory disorders immunologic disorders, cancer IL-1 inhibitor (IL-li) Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of R35486 and R35484 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis EPS541920 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity can be determined using assays known in the art: Matthews et al., in Lymphokines and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Orencole & Dinarclio (1989) inflammatory disorders immunologic disorders, cancer ICE 22 kD subunit. Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of R33780 EP533350 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis SEQ ID NO: 16 of of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity EP533350 can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 450) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225. inflammatory disorders immunologic disorders, cancer ICE 20 kD subunit. Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of R33781 EP533350 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis SEQ ID NO: 17 of of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity EP533350 can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 451) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225. inflammatory disorders immunologic disorders, cancer ICE 10 kD subunit Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of R33782 EP533350 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis SEQ ID NO: 18 of of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity EP533350 can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 452) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225. inflammatory disorders immunologic disorders, cancer Human Interleukin-10 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, (precursor) GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession R41664 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis WO9317698 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity P22301/IL10_HUMAN can be determined using assays known in the art: Matthews et al., in Lymphokines Interleukin-10 and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (precursor form is D.C. 1987, pp. 221-225; and Thompson-Snipes et al (1991) J. Exp. Med. 173, 507-510. processed into a truncated inflammatory disorders immunologic disorders, cancer mature form) (SEQ ID NO: 272) Human Interleukin-10 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of R42642 WO9318783 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis SEQ ID NO: 3 of of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity WO9318783-A can be determined using assays known in the art: Matthews et al., in Lymphokines (mature IL-10) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (SEQ ID NO: 273) D.C. 1987, pp. 221-225; and Thompson-Snipes et al (1991) J. Exp. Med. 173, 507-510. inflammatory disorders immunologic disorders, cancer Human Interleukin-1 beta Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, precursor GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession R42447 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis EP569042 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity P01584/IL1B_HUMAN can be determined using assays known in the art: Matthews et al., in Lymphokines Interleukin-1 beta and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (SEQ ID NO: 274) D.C. 1987, pp. 221-225; Kitamura et al (1989) J Cell Physiol. 140 323-334. inflammatory disorders immunologic disorders, cancer Human interleukin-6 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of R49041 WO9403492 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis P05231/IL6_HUMAN of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Interleukin-6 can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 270) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Aarden et al (1987) Eur. J. Immunol 17, 1411-16. inflammatory disorders immunologic disorders, cancer Mutant Interleukin 6 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, S176R GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession R54990 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis WO9411402 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity S176R variant of wildtype can be determined using assays known in the art: Matthews et al., in Lymphokines IL-6 which has the and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, sequence: D.C. 1987, pp. 221-225; and Aarden et al (1987) Eur. J. Immunol 17, 1411-16. P05231/IL6_HUMAN inflammatory disorders immunologic disorders, cancer Interleukin-6 (SEQ ID NO: 270) Interleukin 6 GeneSeq Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, Accession R55256 monocytes, and macrophages. Known functions include stimulating proliferation of JP06145063 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis P05231/IL6_HUMAN of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Interleukin-6 can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 270) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Aarden et al (1987) Eur. J. Immunol 17, 1411-16. inflammatory disorders immunologic disorders, cancer Interleukin 8 (IL-8) Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, receptor GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession R53932 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis JP06100595 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity GenBank: AAA59159.1 can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 275) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Holmes et al (1991) Science 253, 1278-80. Soluble IL-8 receptor polypep- tides may be useful for inhibiting interleukin activities. Human interleukin-7 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of R59919 U.S. Pat. No. immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis 5,328,988 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity P13232/IL7_HUMAN can be determined using assays known in the art: Matthews et al., in Lymphokines Interleukin-7 and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (isoform 1) D.C. 1987, pp. 221-225; and Park et al (1990) J. Exp. Med. 171, 1073-79. (SEQ ID NO: 276) inflammatory disorders immunologic disorders, cancer IL-3 containing fusion Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, protein. GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession R79342 and immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis R79344 WO9521254 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Fusions of wildtype IL-3 can be determined using assays known in the art: Matthews et al., in Lymphokines which has the sequence: and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, P08700|IL3_HUMAN D.C. 1987, pp. 221-225; Kitamura et al (1989) J Cell Physiol. 140 323-334. Interleukin-3 inflammatory disorders immunologic disorders, cancer (SEQ ID NO: 449) IL-3 mutant proteins Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of R79254, R79255, R79256, immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis R79257, R79258, R79259, of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity R79260, R79261, R79262, can be determined using assays known in the art: Matthews et al., in Lymphokines R79263, R79264, R79265, and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, R79266, R79267, R79268, D.C. 1987, pp. 221-225; and Giri et al (1994) EMBO J. 13 2822-2830. inflammatory R79269, R79270, R79271, disorders immunologic disorders, cancer R79272, R79273, R79274, R79275, R79276, R79277, R79278, R79279, R79280, R79281, R79282, R79283, R79284, and R79285 ZA9402636 Variants of wildtype IL-3 which has the sequence: P08700|IL3_HUMAN Interleukin-3 (SEQ ID NO: 449) IL-12 p40 subunit. Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of R63018 AU9466072 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis P2946/|IL12B_HUMAN of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Interleukin-12 subunit beta can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 277) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225. inflammatory disorders immunologic disorders, cancer AGF GeneSeq Accession Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, R64240 WO9429344 monocytes, and macrophages. Known functions include stimulating proliferation of Q8NI99/ANGL6_HUMAN immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis Angiopoietin-related of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity protein 6 can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 278) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225. inflammatory disorders immunologic disorders, cancer Human interlaukin-12 40 kD Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, subunit GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession R79187 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis WO9519786 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity P2946/|IL12B_HUMAN can be determined using assays known in the art: Matthews et al., in Lymphokines Interleukin-12 subunit beta and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (SEQ ID NO: 277) D.C. 1987, pp. 221-225; and Hori et al (1987), Blood 70, 1069-1078. inflammatory disorders immunologic disorders, cancer Human interleukin-15 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, receptor from clone P1 monocytes, and macrophages. Known functions include stimulating proliferation of GeneSeq Accession immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis R90843 WO9530695 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Q13261|I15RA_HUMAN can be determined using assays known in the art: Matthews et al., in Lymphokines Interleukin-15 receptor and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, subunit alpha D.C. 1987, pp. 221-225; and Giri et al (1994) EMBO J. 13 2822-2830. Soluble IL-8 Isoform 1) receptor polypep- tides may be useful for inhibiting interleukin activities. Obesity. (SEQ ID NO: 453) Metabolic Disease. Diabetes. Enhancing secretion and stability of Interleukin 15 Human interleukin-7 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of R92796 WO9604306 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis P13232/IL7_HUMAN of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Interleukin-7 can be determined using assays known in the art: Matthews et al., in Lymphokines (isoform 1) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (SEQ ID NO: 276) D.C. 1987, pp. 221-225; and Park et al (1990) J. Exp. Med. 171, 1073-79. inflammatory disorders immunologic disorders, cancer interleukin-9 GeneSeq Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, Accession R92797 monocytes, and macrophages. Known functions include stimulating proliferation of WO9604306 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis P15248/IL9_HUMAN of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Interleukin-9 can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 279) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Yang et al (1989) Blood 74, 1880-84. inflammatory disorders immunologic disorders, cancer interleukin-3 GeneSeq Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, Accession R92801 monocytes, and macrophages. Known functions include stimulating proliferation of WO9604306 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis P08700|IL3_HUMAN of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Interleukin-3 can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 280) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; Kitamura et al (1989) J Cell Physiol. 140 323-334. inflammatory disorders immunologic disorders, cancer Human interleukin-5 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of R92802 WO9604306 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis P05113/IL5_HUMAN of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Interleukin-5 can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 281) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; Kitamura et al (1989) J Cell Physiol. 140 323-334. inflammatory disorders immunologic disorders, cancer Recombinant interleukin- Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, 16 GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of W33373 DE19617202 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis Q14005/IL16_HUMAN of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Pro-interleukin-16 can be determined using assays known in the art: Matthews et al., in Lymphokines (isoform 1) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (SEQ ID NO: 282) D.C. 1987, pp. 221-225; and Lim et al (1996) J. Immunol. 156, 2566-70. inflammatory disorders immunologic disorders, cancer Human IL-16 protein Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of W33234 DE19617202 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis Q14005/IL16_HUMAN of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Pro-interleukin-16 can be determined using assays known in the art: Matthews et al., in Lymphokines (isoform 1) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (SEQ ID NO: 282) D.C. 1987, pp. 221-225; and Lim et al (1996) J. Immunol. 156, 2566-70. inflammatory disorders immunologic disorders, cancer Thrl 17 human interleukin Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, 9 GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of W27521 WO9708321 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis P15248|IL9_HUMAN of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Interleukin-9 can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 387) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225. inflammatory disorders immunologic disorders, cancer Metl 17 human interleukin Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, 9 GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of W27522 WO9708321 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis (SEQ ID NO: 388) of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity can be determined using assays known in the art: Matthews et al., in Lymphokines and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Yang et al (1989) Blood 74, 1880-84. inflammatory disorders immunologic disorders, cancer Human intracellular IL-1 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, receptor antagonist. monocytes, and macrophages. Known functions include stimulating proliferation of GeneSeq Accession immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis W77158 EP864585 (e.g. of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity SEQ ID NOs: 12 to 19, or can be determined using assays known in the art: Matthews et al., in Lymphokines 22 to 25 of this and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, publication). D.C. 1987, pp. 221-225; and Orencole & Dinarello (1989) Cytokine 1, 14-20. inflammatory disorders immunologic disorders, cancer Human interleukin-18 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, protein (IL-18) GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession W77158 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis EP864585 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Q14116/IL18_HUMAN can be determined using assays known in the art: Matthews et al., in Lymphokines Interleukin-18 and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (isoform 1) D.C. 1987, pp. 221-225; and USHIO et al (1996) J. Immunol. 156, 4274-79. (SEQ ID NO: 283) inflammatory disorders immunologic disorders, cancer Human interleukin-18 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of W77077 EP861663 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis Q14116/IL18_HUMAN of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Interleukin-18 can be determined using assays known in the art: Matthews et al., in Lymphokines (isoform 1) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (SEQ ID NO: 283) D.C. 1987, pp. 221-225; and USHIO et al (1996) J. Immunol. 156, 4274-79. inflammatory disorders immunologic disorders, cancer Human interleukin 18 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, derivatives GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accessions W77083, immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis W77084, W77085, of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity W77086, W77087, can be determined using assays known in the art: Matthews et al., in Lymphokines W77088, and W77089 and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, EP861663 D.C. 1987, pp. 221-225; and Ushio et al (1996) J. Immunol, 156, 4274-79. Variants of wildtype IL18 inflammatory disorders immunologic disorders, cancer which is provided as: Q14116/IL18_HUMAN Interleukin-18 (isoform 1) (SEQ ID NO: 283) Interleukin-9 (IL-9) mature Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, protein (Thr117 version). monocytes, and macrophages. Known functions include stimulating proliferation of GeneSeq Accession immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis W68158 WO9827997 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity FIG. 2 of WO9827997 can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 389) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Yang et al (1989) Blood 74, 1880-84. inflammatory disorders immunologic disorders, cancer IL-9 mature protein variant Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, (Met117 version) GenSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession W68157 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis WO9827997 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity FIG. 3 of WO9827997 can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 390) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Yang et al (1989) Blood 74, 1880-84. inflammatory disorders immunologic disorders, cancer Human IL-9 receptor Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, protein variant #3. monocytes, and macrophages. Known functions include stimulating proliferation of GeneSeq Accession immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis W64058 WO9824904 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Wildtype IL-9R is provided can be determined using assays known in the art: Matthews et al., in Lymphokines as: and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, Q01113/IL9R_HUMAN D.C. 1987, pp. 221-225; and Yang et al (1989) Blood 74, 1880-84. inflammatory Interleukin-9 receptor disorders immunologic disorders, cancer (isoform 1) (SEQ ID NO: 303) Human IL-9 receptor Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, protein variant fragment monocytes, and macrophages. Known functions include stimulating proliferation of GenSeq Accession immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis W64060 WO9824904 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Wildtype IL-9R is provided can be determined using assays known in the art: Matthews et al., in Lymphokines as: and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, Q01113/IL9R_HUMAN D.C. 1987, pp. 221-225; and Yang et al (1989) Blood 74, 1880-84. Soluble IL-9 Interleukin-9 receptor receptor polypep- tides may be useful for inhibiting interleukin activities. (isoform 1) (SEQ ID NO: 303) Human IL-9 receptor Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, protein variant #3. monocytes, and macrophages. Known functions include stimulating proliferation of GeneSeq Accession immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis W64061 WO9824904 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Wildtype IL-9R is provided can be determined using assays known in the art: Matthews et al., in Lymphokines as: and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, Q01113/IL9R_HUMAN D.C. 1987, pp. 221-225; and Yang et al (1989) Blood 74, 1880-84. Soluble IL-9 Interleukin-9 receptor receptor polypep- tides may be useful for inhibiting interleukin activities. (isoform 1) (SEQ ID NO: 303) Human Interleukin-12 p40 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, protein GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession W51311 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis WO9817689 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity P2946/|IL12B_HUMAN can be determined using assays known in the art: Matthews et al., in Lymphokines Interleukin-12 subunit beta and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (SEQ ID NO: 277) D.C. 1987, pp. 221-225; and Hori et al (1987), Blood 70, 1069-1078. inflammatory disorders immunologic disorders, cancer Human Interleukin-12 p35 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, protein GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession W51312 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis WO9817689 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity P29459/IL12A_HUMAN can be determined using assays known in the art: Matthews et al., in Lymphokines Interleukin-12 subunit and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, alpha D.C. 1987, pp. 221-225; and Hori et al (1987), Blood 70, 1069-1078. inflammatory (SEQ ID NO: 284) disorders immunologic disorders, cancer Human protein with IL-16 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, activity GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession W63753 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis DE19649233- of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity can be determined using assays known in the art: Matthews et al., in Lymphokines and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Lim et al (1996) J. Immunol. 156, 2566-70. inflammatory disorders immunologic disorders, cancer Human protein with IL-16 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, activity GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession W59425 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis DE19649233- of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity can be determined using assays known in the art: Matthews et al., in Lymphokines and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Lim et al (1996) J. Immunol. 156, 2566-70. inflammatory disorders immunologic disorders, cancer Human interleukin-15 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of W53878 U.S. Pat. No. immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis 5,747,024 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity P40933/IL15_HUMAN can be determined using assays known in the art: Matthews et al., in Lymphokines Interleukin-15 and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (isoform IL15-S48AA) D.C. 1987, pp. 221-225; and Giri et al (1994) EMBO J. 13 2822-2830. inflammatory (SEQ ID NO: 285) disorders immunologic disorders, cancer. Obesity. Metabolic Disease. Diabetes. Human wild-type Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, interleukin-4 (hIL-4) monocytes, and macrophages. Known functions include stimulating proliferation of protein GeneSeq immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis Accession W52149 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity WO9747744 can be determined using assays known in the art: Matthews et al., in Lymphokines P05112/IL4_HUMAN and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, Interleukin-4 D.C. 1987, pp. 221-225; and Siegel & Mostowski (1990) J Immunol Methods 132, (isoform 1) 287-295. inflammatory disorders immunologic disorders, cancer (SEQ ID NO: 286) interleukin-4 muteins Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, GeneSeq Accessions monocytes, and macrophages. Known functions include stimulating proliferation of W52150, W52151, immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis W52153, W52154, of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity W52155, W52156, can be determined using assays known in the art: Matthews et al., in Lymphokines W52157, W52158, and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, W52159, W52160, D.C. 1987, pp. 221-225; and Siegel & Mostowski (1990) J Immunol Methods 132, W52161, W52162, 287-295. inflammatory disorders immunologic disorders, cancer W52163, W52164, W52165, W52166, and W52167 WO9747744 Variants of wildtype IL-4 which has the sequence: P05112/IL4_HUMAN Interleukin-4 (isoform 1) (SEQ ID NO: 268) Human interleukin 1 delta Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of Y28408 WO9935268 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis SEQ ID NO: 4 of of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity WO9935268 can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 287) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Orencole & Dinarello (1989) Cytokine 1, 14-20. inflammatory disorders immunologic disorders, cancer Human interleukin-1 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, receptor antagonist beta monocytes, and macrophages. Known functions include stimulating proliferation of GeneSeq Accession immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis Y24395 WO9935268 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity can be determined using assays known in the art: Matthews et al., in Lymphokines and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Orencole & Dinarello (1989) Cytokine 1, 14-20. inflammatory disorders immunologic disorders, cancer Human EDIRF II protein Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, sequence GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession Y22199 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis WO9932632 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity SEQ ID NO: 6 of can be determined using assays known in the art: Matthews et al., in Lymphokines WO9932632 and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (SEQ ID NO: 391) D.C. 1987, pp. 221-225. inflammatory disorders immunologic disorders, cancer Human EDIRF I protein Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, sequence GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession Y22197 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis WO9932632 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity SEQ ID NO: 2 of can be determined using assays known in the art: Matthews et al., in Lymphokines WO9932632 and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (SEQ ID NO: 392) D.C. 1987, pp. 221-225. inflammatory disorders immunologic disorders, cancer Human IL- 1RD10 protein Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, sequence GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession Y14131 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis WO9919480 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity SEQ ID NO: 20 of can be determined using assays known in the art: Matthews et al., in Lymphokines WO9919480 and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Orencole & Dinarello (1989) Cytokine 1, 14-20. Soluble IL-1RD10 receptor polypep- tides may be useful for inhibiting interleukin activites. Human IL- 1RD9 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of Y14122 WO9919480 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis SEQ ID NOS: 6, 8, 10 of of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity WO9919480 can be determined using assays known in the art: Matthews et al., in Lymphokines and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Orencole & Dinarello (1989) Cytokine 1, 14-20. Soluble IL-1RD10 receptor polypep- tides may be useful for inhibiting interleukin activites. Human DNAX interleukin- Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, 40 GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of Y09196 WO9919491 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis SEQ ID NO: 2 or 4 of of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity WO9919491 can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 454) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225. inflammatory disorders immunologic disorders, cancer (DIL-40) alternative Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, sequence GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession Y09197 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis WO9919491 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity SEQ ID NO: 4 of can be determined using assays known in the art: Matthews et al., in Lymphokines WO9919491 and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (SEQ ID NO: 455) D.C. 1987, pp. 221-225. inflammatory disorders immunologic disorders, cancer IL-11 GeneSeq Accession Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, R50176 WO9405318 monocytes, and macrophages. Known functions include stimulating proliferation of P2080/|IL11_HUMAN immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis Interleukin-11 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity (isoform 1) can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 288) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Lu et al (1994) J immunol. Methods 173, 19. inflammatory disorders immunologic disorders, cancer Human adipogenesis Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, inhibitory factor GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession R43260 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis EP566410 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity (also known as IL-11) can be determined using assays known in the art: Matthews et al., in Lymphokines P2080/|IL11_HUMAN and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, Interleukin-11 D.C. 1987, pp. 221-225. inflammatory disorders immunologic disorders, cancer (isoform 1) (SEQ ID NO: 288) IL-11 GeneSeq Accession Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, W02202 JP08127539 monocytes, and macrophages. Known functions include stimulating proliferation of P2080/|IL11_HUMAN immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis Interleukin-11 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity (isoform 1) can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 288) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Lu et al (1994) J immunol. Methods 173, 19. inflammatory disorders immunologic disorders, cancer IL-14 GeneSeq Accession Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, R55800 WO9416074 monocytes, and macrophages. Known functions include stimulating proliferation of P40222/TXLNA_HUMAN immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis Alpha-taxilin of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity (SEQ ID NO: 289) can be determined using assays known in the art: Matthews et al., in Lymphokines and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Ambrus et al (1993) PNAS 90, 63330-34. inflammatory disorders immunologic disorders, cancer IL-17 receptor GeneSeq Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, Accession B03807 U.S. monocytes, and macrophages. Known functions include stimulating proliferation of Pat. No. 6,072,033 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis Q96F46/I17RA_HUMAN of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Interleukin-17 receptor A can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 290) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Yao et al (1995) J. Immunol. 155, 5483-86. Soluble IL- 17 receptor polypep- tides may be useful for inhibiting interleukin activites. IL-17 GeneSeq Accession Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, R76573 WO9518826 monocytes, and macrophages. Known functions include stimulating proliferation of Q16552/IL17_HUMAN immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis Interleukin-17A of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity (SEQ ID NO: 291) can be determined using assays known in the art: Matthews et al., in Lymphokines and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Yao et al (1995) J. Immunol. 155, 5483-86. inflammatory disorders immunologic disorders, cancer CTLA-8 GeneSeq Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, Accession W13651 monocytes, and macrophages. Known functions include stimulating proliferation of WO9704097 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis (also known as IL-17) of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Q16552/IL17_HUMAN can be determined using assays known in the art: Matthews et al., in Lymphokines Interleukin-17A and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (SEQ ID NO: 291) D.C. 1987, pp. 221-225. inflammatory disorders immunologic disorders, cancer IL-19 GeneSeq Accession Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, W37935 WO9808870 monocytes, and macrophages. Known functions include stimulating proliferation of Q9UHD0|IL19_HUMAN immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis Interleukin-19 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity (isoform 1) can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 292) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Gallagher et al (2000) Genes Immun. 1,442-50. inflammatory disorders immunologic disorders, cancer IL-21 (TIF) GeneSeq Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, Accession Y92879 monocytes, and macrophages. Known functions include stimulating proliferation of WO0024758 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis Q9HBE4/IL21_HUMAN of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Interleukin-21 can be determined using assays known in the art: Matthews et al., in Lymphokines (isoform 1) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (SEQ ID NO: 293) D.C. 1987, pp. 221-225; and Parrish-Novak et al (2000) Nature 408, 57-63. inflammatory disorders immunologic disorders, cancer IL-8 receptor GeneSeq Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, Accession R33420 monocytes, and macrophages. Known functions include stimulating proliferation of WO9306229 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis IL-8RA of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity P25024/CXCR1_HUMAN can be determined using assays known in the art: Matthews et al., in Lymphokines C-X-C chemokine receptor and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, type 1 D.C. 1987, pp. 221-225; and Holmes et al (1991) Science 253, 1278-80. Soluble IL-8 (SEQ ID NO: 294) receptor polypep- tides may be useful for inhibiting interleukin activities. IL-8RB P25025/CXCR2_HUMAN C-X-C chemokine receptor type 2 (SEQ ID NO: 295) Human type II interleukin- Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, 1 receptor GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession R85480 U.S. immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis Pat. No. 5,464,937 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity P27930/IL1R2_HUMAN can be determined using assays known in the art: Matthews et al., in Lymphokines Interleukin-1 receptor type 2 and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (SEQ ID NO: 296) D.C. 1987, pp. 221-225; and Orencole & Dinarello (1989) Cytokine 1, 14-20. Soluble type II interleukin-1 receptor polypep- tides may be useful for inhibiting interleukin activities. Human interleukin-12 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, receptor GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession R69632 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis EP638644 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity IL-12 receptor B1 can be determined using assays known in the art: Matthews et al., in Lymphokines P42701|I12R1_HUMAN and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, Interleukin-12 receptor D.C. 1987, pp. 221-225; and Hori et al (1987), Blood 70, 1069-1078. Soluble IL-12 subunit beta-1 receptor polypep- tides may be useful for inhibiting interleukin activities. (isoform 1) (SEQ ID NO: 393) IL-12 receptor B2 Q99665|I12R2_HUMAN Interleukin-12 receptor subunit beta-2 (isoform 1) (SEQ ID NO: 394) Interleukin 8 receptor B Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of R80758 U.S. Pat. No. immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis 5,440,021 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity IL-8RB can be determined using assays known in the art: Matthews et al., in Lymphokines P25025/CXCR2_HUMAN and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, C-X-C chemokine receptor D.C. 1987, pp. 221-225; and Holmes et al (1991) Science 253, 1278-80. Soluble IL-8 type 2 receptor B polypep- tides may be useful for inhibiting interleukin activities. (SEQ ID NO: 295) Human IL-8 receptor Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, protein hIL8RA GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession B09989 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis JP08103276 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity IL-8RA can be determined using assays known in the art: Matthews et al., in Lymphokines P25024/CXCR1_HUMAN and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, C-X-C chemokine receptor D.C. 1987, pp. 221-225; and Holmes et al (1991) Science 253, 1278-80. Soluble IL-8 type 1 receptor A polypep- tides may be useful for inhibiting interleukin activities. (SEQ ID NO: 294) Human IL-8 receptor Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, protein hIL8R GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession B09990 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis JP08103276 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity IL-8RA can be determined using assays known in the art: Matthews et al., in Lymphokines P25024/CXCR1_HUMAN and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, C-X-C chemokine receptor D.C. 1987, pp. 221-225; and Holmes et al (1991) Science 253, 1278-80. Soluble IL-8 type 1 receptor polypep- tides may be useful for inhibiting interleukin activities. (SEQ ID NO: 294) IL-8RB P25025/CXCR2_HUMAN C-X-C chemokine receptor type 2 (SEQ ID NO: 295) Interleukin-2 receptor Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, associated protein p43 monocytes, and macrophages. Known functions include stimulating proliferation of GeneSeq Accession immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis R97569 WO9621732- of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity SEQ ID NO: 2 of can be determined using assays known in the art: Matthews et al., in Lymphokines WO9621732 and Interferons: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (SEQ ID NO: 395) D.C. 1987, pp. 221-225; and Gillis et al (1978) J. Immunol. 120, 2027. Soluble IL-2 receptor polypep- tides may be useful for inhibiting interleukin activities. Human interleukin-17 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, receptor GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession W04185 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis WO9629408 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Q96F46/I17RA_HUMAN can be determined using assays known in the art: Matthews et al., in Lymphokines Interleukin-17 receptor A and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (SEQ ID NO: 290) D.C. 1987, pp. 221-225; and Yao et al (1995) J. Immunol. 155, 5483-86. Soluble IL- 17 receptor polypep- tides may be useful for inhibiting interleukin activities. Human interleukin-11 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, receptor GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession R99090 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis WO9619574 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Q14626/I11RA_HUMAN can be determined using assays known in the art: Matthews et al., in Lymphokines Interleukin-11 receptor and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, subunit alpha D.C. 1987, pp. 221-225; and Lu et al (1994) J immunol. Methods 173, 19. Soluble IL- (SEQ ID NO: 297) 11 receptor polypep- tides may be useful for inhibiting interleukin activities. Human interleukin-1 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, receptor accessory protein monocytes, and macrophages. Known functions include stimulating proliferation of GeneSeq Accession immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis W01911 WO9623067 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Human IL1R Acp can be determined using assays known in the art: Matthews et al., in Lymphokines SEQ ID NO: 3 of and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, WO9623067 D.C. 1987, pp. 221-225; and Orencole & Dinarello (1989) Cytokine 1, 14-20. (SEQ ID NO: 396) Inflammatory disorders immunologic disorders, cancer Soluble Human IL1R Acp SEQ ID NO: 9 of WO9623067 (SEQ ID NO: 397) AGF Protein GeneSeq Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, Accession R92749 U.S. monocytes, and macrophages. Known functions include stimulating proliferation of Pat. No. 5,488,032 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis Q8NI99/ANGL6_HUMAN of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Angiopoietin-related can be determined using assays known in the art: Matthews et al., in Lymphokines protein 6 and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (SEQ ID NO: 278) D.C. 1987, pp. 221-225. Inflammatory disorders immunologic disorders, cancer Human interleukin-1 type- Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, 3 receptor GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession R91064 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis W09607739 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity SEQ ID NO: 2 and 4 of can be determined using assays known in the art: Matthews et al., in Lymphokines WO9607739 and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (SEQ ID NO: 398 and SEQ D.C. 1987, pp. 221-225; and Orencole & Dinarello (1989) Cytokine 1, 14-20. Soluble ID NO: 399, respectively) IL-type-3 receptor polypep- tides may be useful for inhibiting interleukin activities Human interleukin-13 beta Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, receptor GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession W24972 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis WO9720926 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity SEQ ID NO: 2 from can be determined using assays known in the art: Matthews et al., in Lymphokines WO9720926 and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (SEQ ID NO: 400) D.C. 1987, pp. 221-225; and Boutelier et al (1995) J. Immunol. Methods 181, 29. Soluble IL-13 beta receptor polypep- tides may be useful for inhibiting interleukin activities. Human interleukin-13 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, alpha receptor GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession W24973 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis WO9720926 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity IL-13RA1 can be determined using assays known in the art: Matthews et al., in Lymphokines P78552/I13R1_HUMAN and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, Interleukin-13 receptor D.C. 1987, pp. 221-225; and Boutelier et al (1995) J. Immunol. Methods 181, 29. subunit alpha-1 Soluble IL-13 alpha receptor polypep- tides may be useful for inhibiting interleukin (isoform 1) activities. (SEQ ID NO: 298) IL-13RA2 Q14627/I13R2_HUMAN Interleukin-13 receptor subunit alpha-2 (SEQ ID NO: 299) Human interleukin-4 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, receptor GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession W13499 U.S. immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis Pat. No. 5,599,905 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity P24394/IL4RA_HUMAN can be determined using assays known in the art: Matthews et al., in Lymphokines Interleukin-4 receptor and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, subunit alpha D.C. 1987, pp. 221-225; and Siegel & Mostowski (1990) J Immunol Methods 132, (isoform 1) 287-295. Soluble IL-4 receptor polypep- tides may be useful for inhibiting interleukin (SEQ ID NO: 300) activities. Human interleukin-12 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, beta-2 receptor GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession W12771 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis EP759466 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Q9966/|I12R2_HUMAN can be determined using assays known in the art: Matthews et al., in Lymphokines Interleukin-12 receptor and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, subunit beta-2 D.C. 1987, pp. 221-225; and Hori et al (1987), Blood 70, 1069-1078. Soluble IL-12 (isoform 1) beta-2 receptor polypep- tides may be useful for inhibiting interleukin activities. (SEQ ID NO: 301) Human interleukin-12 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, beta-1 receptor. GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession W12772 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis EP759466 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity P4270/|I12R1_HUMAN can be determined using assays known in the art: Matthews et al., in Lymphokines Interleukin-12 receptor and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, subunit beta-1 D.C. 1987, pp. 221-225; and Hori et al (1987), Blood 70, 1069-1078. Soluble IL-12 (isoform 1) beta-1 receptor polypep- tides may be useful for inhibiting interleukin activities. (SEQ ID NO: 302) Human IL-9 receptor Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, protein GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accessions W64055, immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis W64056, and W64057 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity WO9824904 can be determined using assays known in the art: Matthews et al., in Lymphokines Q01113/IL9R_HUMAN and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, Interleukin-9 receptor D.C. 1987, pp. 221-225; and Yang et al (1989) Blood 74, 1880-84. Soluble IL-9 (isoform 1) receptor polypep- tides may be useful for inhibiting interleukin activities. (SEQ ID NO: 303) IL-10 receptor GeneSeq Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, Accession W41804 U.S. monocytes, and macrophages. Known functions include stimulating proliferation of Pat. No. 5,716,804 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis IL-10RA of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Q13651/I10R1_HUMAN can be determined using assays known in the art: Matthews et al., in Lymphokines Interleukin-10 receptor and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, subunit alpha D.C. 1987, pp. 221-225; and Thompson-Snipes et al (1991) J. Exp. Med. 173, 507-510. (SEQ ID NO: 304) Soluble IL-10 receptor polypep- tides may be useful for inhibiting interleukin IL-10RB activities. Q0833/|I10R2_HUMAN Interleukin-10 receptor subunit beta (SEQ ID NO: 305) Human IL-6 receptor Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of Y30938 JP11196867 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis P08887/IL6RA_HUMAN of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Interleukin-6 receptor can be determined using assays known in the art: Matthews et al., in Lymphokines subunit alpha and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (isoform 1) D.C. 1987, pp. 221-225; and Aarden et al (1987) Eur. J. Immunol 17, 1411-16. (SEQ ID NO: 306) Soluble IL-6 receptor polypep- tides may be useful for inhibiting interleukin activities. II-17 receptor GeneSeq Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, Accession Y97181 U.S. monocytes, and macrophages. Known functions include stimulating proliferation of Pat. No. 6,096,305 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis Q96F46/I17RA_HUMAN of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Interleukin-17 receptor A can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 290) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Yao et al (1995) J. Immunol. 155, 5483-86. Soluble IL- 17 receptor polypep- tides may be useful for inhibiting interleukin activities. II-17 receptor GeneSeq Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, Accession Y97131 U.S. monocytes, and macrophages. Known functions include stimulating proliferation of Pat. No. 6,100,235 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis Q96F46/I17RA_HUMAN of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Interleukin-17 receptor A can be determined using assays known in the art: Matthews et al., in Lymphokines (SEQ ID NO: 290) and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, D.C. 1987, pp. 221-225; and Yao et al (1995) J. Immunol. 155, 5483-86. Soluble IL- 17 receptor polypep- tides may be useful for inhibiting interleukin activities. Human interleukin-3 Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, receptor GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession R25300 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis EP509826 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity P26951/IL3RA_HUMAN can be determined using assays known in the art: Matthews et al., in Lymphokines Interleukin-3 receptor and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, subunit alpha D.C. 1987, pp. 221-225; Kitamura et al (1989) J Cell Physiol. 140 323-334. Soluble (isoform 1) IL-3 receptor polypep- tides may be useful for inhibiting interleukin activities. (SEQ ID NO: 307) Human GM- CSF receptor Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of R10919 WO9102063 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis GM-CSF receptor A of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity P15509/CSF2R_HUMAN can be determined using assays known in the art: Matthews et al., in Lymphokines Granulocyte-macrophage and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, colony-stimulating factor D.C. 1987, pp. 221-225. Soluble GM-CSF receptor polypep- tides may be useful for receptor subunit alpha inhibiting interleukin activities. (isoform 1) (SEQ ID NO: 308) GM-CSF receptor B P32927/IL3RB_HUMAN Cytokine receptor common subunit beta (isoform 1) (SEQ ID NO: 309) Human IL-5 receptor alpha Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, chain GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of R25064 EP492214 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis Q01344/IL5RA_HUMAN of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Interleukin-5 receptor can be determined using assays known in the art: Matthews et al., in Lymphokines subunit alpha and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (isoform 1) D.C. 1987, pp. 221-225; Kitamura et al (1989) J Cell Physiol. 140 323-334. Soluble (SEQ ID NO: 310) IL-5 receptor alpha polypeptides may be useful for inhibiting interleukin activities. II-5 receptor GeneSeq Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, Accession W82842 monocytes, and macrophages. Known functions include stimulating proliferation of WO9847923 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis Q01344/IL5RA_HUMAN of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Interleukin-5 receptor can be determined using assays known in the art: Matthews et al., in Lymphokines subunit alpha and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (isoform 1) D.C. 1987, pp. 221-225; Kitamura et al (1989) J Cell Physiol. 140 323-334. Soluble (SEQ ID NO: 310) IL-5 receptor polypep- tides may be useful for inhibiting interleukin activities. II-6 receptor GeneSeq Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, Accession R37215 monocytes, and macrophages. Known functions include stimulating proliferation of JP05091892 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis P08887/IL6RA_HUMAN of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Interleukin-6 receptor can be determined using assays known in the art: Matthews et al., in Lymphokines subunit alpha and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (isoform 1) D.C. 1987, pp. 221-225; and Aarden et al (1987) Eur. J. Immunol 17, 1411-16. (SEQ ID NO: 306) Soluble IL-6 receptor polypep- tides may be useful for inhibiting interleukin activities. Human B cell stimulating Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, factor-2 receptor GeneSeq monocytes, and macrophages. Known functions include stimulating proliferation of Accession P90525 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis AU8928720 of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity P08887/IL6RA HUMAN can be determined using assays known in the art: Matthews et al., in Lymphokines Interleukin-6 receptor and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, subunit alpha D.C. 1987, pp. 221-225. Soluble B cell stimulating factor-2 receptor polypep- tides (isoform 1) may be useful for inhibiting interleukin activities. (SEQ ID NO: 306) IL-7 receptor clone Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, GeneSeq Accession monocytes, and macrophages. Known functions include stimulating proliferation of R08330 EP403114 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis P1687/|IL7RA_HUMAN of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Interleukin-7 receptor can be determined using assays known in the art: Matthews et al., in Lymphokines subunit alpha and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (isoform 1) D.C. 1987, pp. 221-225; and Park et al (1990) J. Exp. Med. 171, 1073-79. Soluble IL- (SEQ ID NO: 311) 7 receptor polypep- tides may be useful for inhibiting interleukin activities. EPO receptor; EPOR EPO Receptor is involved in the proliferation and differentiation of erythroblasts. EPO GeneSeq Accession Receptor activity can be determined using assays known in the art, such as, J Biol R06512 WO9008822 Chem 2001 Mar. 23; 276(12: 8995-9002; JAK2 protein tyrosine kinase activity: Blood P19235/EPOR_HUMAN 1994 Sep. 1; 84(5): 1501-7 and Mol Cell Biol. 1994 October; 14(10: 6506-14. Inflammatory Erythropoietin receptor disorders, immunologic disorders, cancer, erythroblast proliferation and differentiation (isoform EPOR-F) (SEQ ID NO: 312) IL-15 receptor GeneSeq Interleukins are a group of multi- functional cytokines synthesized by lymphocytes, Accession R90843 monocytes, and macrophages. Known functions include stimulating proliferation of WO9530695 immune cells (e.g., T helper cells, B cells, eosinophils, and lymphocytes), chemotaxis Q1326/|I15RA_HUMAN of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity Interleukin-15 receptor can be determined using assays known in the art: Matthews et al., in Lymphokines subunit alpha and Interferens: A Practical Approach, Clemens et al., eds, IRL Press, Washington, (isoform 1) D.C. 1987, pp. 221-225; and Giri et al (1994) EMBO J. 13 2822-2830. Soluble IL-15 (SEQ ID NO: 313) receptor polypep- tides may be useful for inhibiting interleukin activities. Obesity. Metabolic Disease. Diabetes. Enhancing secretion and stability of Interleukin 15 CD137; 4-1BB Receptor Activities associated with apoptosis, NF-kB activation, and co- stimulation of immune Protein GeneSeq cells such as T and B cells. Apoptosis activity, NF-kB activation, and B and T cell co- Accession R70977 stimulation can be determined using assays known in the art: Moore et al., 1999, WO9507984 Science, 285(5425): 260-3; Song H Y et al., 1997 Proc Natl Acad Sci USA 94(18): Q07011/TNR9_HUMAN 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Soluble 4-1BB Tumor necrosis factor receptor polypeptides may be useful for inhibiting apoptosis, NF-kB activation, receptor superfamily and/or co-stimulation of immune cells such as B and T cells. member 9 (SEQ ID NO: 314) BCMA GeneSeq Activities associated with apoptosis, NF-kB activation, and co- stimulation of immune Accession Y71979 cells such as T and B cells. Apoptosis activity, NF-kB activation, and B and T cell co- WO0068378 stimulation can be determined using assays known in the art: Moore et al., 1999, Q02223/TNR17_HUMAN Science, 285(5425): 260-3; Song H Y et al., 1997 Proc Natl Acad Sci USA 94(18): Tumor necrosis factor 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Soluble BCMA receptor superfamily receptor polypeptides may be useful for inhibiting apoptosis, NF-kB activation, member 17 and/or co-stimulation of immune cells such as B and T cells. (isoform 1) (SEQ ID NO: 315) CD27 GeneSeq Accession Activities associated with apoptosis, NF-kB activation, and co- stimulation of immune R20814 WO9201049 cells such as T and B cells. Apoptosis activity, NF-kB activation, and B and T cell co- P26842/CD27_HUMAN stimulation can be determined using assays known in the art: Moore et al., 1999, CD27 antigen Science, 285(5425): 260-3; Song H Y et al., 1997 Proc Natl Acad Sci USA 94(18): (SEQ ID NO: 316) 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Soluble CD27 polypeptides may be useful for inhibiting apoptosis, NF-kB activation, and/or co- stimulation of immune cells such as B and T cells. CD30 GeneSeq Accession Activities associated with apoptosis, NF-kB activation, and co- stimulation of immune R35478 DE4200043 cells such as T and B cells. Apoptosis activity, NF-kB activation, and B and T cell co- P28908/TNR8_HUMAN stimulation can be determined using assays known in the art: Moore et al., 1999, Tumor necrosis factor Science, 285(5425): 260-3; Song H Y et al., 1997 Proc Natl Acad Sci USA 94(18): receptor superfamily 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Soluble CD30 member 8 polypeptides may be useful for inhibiting apoptosis, NF-kB activation, and/or co- (isoform 1) stimulation of immune cells such as B and T cells. (SEQ ID NO: 317) CD40 GeneSeq Accession Activities associated with apoptosis, NF-kB activation, and co- stimulation of immune Y33499 WO9945944 cells such as T and B cells. Apoptosis activity, NF-kB activation, and B and T cell co- P25942/TNR5_HUMAN stimulation can be determined using assays known in the art: Moore et al., 1999, Tumor necrosis factor Science, 285(5425): 260-3; Song H Y et al., 1997 Proc Natl Acad Sci USA 94(18): receptor superfamily 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Soluble CD40 member 5 polypeptides may be useful for inhibiting apoptosis, NF-kB activation, and/or co- (isoform 1) stimulation of immune cells such as B and T cells. (SEQ ID NO: 318) EDAR Genbank Activities associated with apoptosis, NF-kB activation, and co- stimulation of immune Accession AAD50077 cells such as T and B cells. Apoptosis activity, NF-kB activation, and B and T cell co- Q9UNE0|EDAR_HUMAN stimulation can be determined using assays known in the art: Moore et al., 1999, Tumor necrosis factor Science, 285(5425): 260-3; Song H Y et al., 1997 Proc Natl Acad Sci USA 94(18): receptor superfamily 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Immune Disorders, member EDAR Lymphomas, X-linked hypohidrotic ectodermal dysplasia (isoform 1) (SEQ ID NO: 319) OX40; ACT-4 GeneSeq Activities associated with apoptosis, NF-kB activation, and co- stimulation of immune Accession R74737 cells such as T and B cells. Apoptosis activity, NF-kB activation, and B and T cell co- WO9512673 stimulation can be determined using assays known in the art: Moore et al., 1999, P43489/TNR4_HUMAN Science, 285(5425): 260-3; Song H Y et al., 1997 Proc Natl Acad Sci USA 94(18): Tumor necrosis factor 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Immune Disorders, receptor superfamily Lymphomas, T cell disorders member 4 (SEQ ID NO: 320) TACI GeneSeq Accession Activities associated with apoptosis, NF-kB activation, and co- stimulation of immune W75783 WO9839361 cells such as T and B cells. Apoptosis activity, NF-kB activation, and B and T cell co- O14836/TR13B_HUMAN stimulation can be determined using assays known in the art: Moore et al., 1999, Tumor necrosis factor Science, 285(5425): 260-3; Song H Y et al., 1997 Proc Natl Acad Sci USA 94(18): receptor superfamily 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Soluble TACI member 13B receptor polypep- tides may be useful for inhibiting apoptosis, NF-kB activation, (isoform 1) and/or co-stimulation of immune cells such as B and T cells. (SEQ ID NO: 321) TNF-R GeneSeq Activities associated with apoptosis, NF-kB activation, and co- stimulation of immune Accession R10986 cells such as T and B cells. Apoptosis activity, NF-kB activation, and B and T cell co- AU9058976 stimulation can be determined using assays known in the art: Moore et al., 1999, P19438/TNR1A_HUMAN Science, 285(5425): 260-3; Song H Y et al., 1997 Proc Natl Acad Sci USA 94(18): Tumor necrosis factor 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Soluble TNF-R receptor superfamily receptor polypep- tides may be useful for inhibiting apoptosis, NF-kB activation, member 1A and/or co-stimulation of immune cells such as B and T cells. (isoform 1) (SEQ ID NO: 322) TNF-RII; TNF p75 Activities associated with apoptosis, NF-kB activation, and co- stimulation of immune receptor; Death Receptor cells such as T and B cells. Apoptosis activity, NF-kB activation, and B and T cell co- GeneSeq Accession stimulation can be determined using assays known in the art: Moore et al., 1999, R11141 EP418014 Science, 285(5425): 260-3; Song H Y et al., 1997 Proc Natl Acad Sci USA 94(18): P20333/TNR1B_HUMAN 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Soluble TNFR-II Tumor necrosis factor receptor polypep- tides may be useful for inhibiting apoptosis, NF-kB activation, receptor superfamily and/or co-stimulation of immune cells such as B and T cells. member 1B (isoform 1) (SEQ ID NO: 323) hAPO-4; TROY GeneSeq Activities associated with apoptosis, NF-kB activation, and co- stimulation of immune Accession W93581 cells such as T and B cells. Apoptosis activity, NF-kB activation, and B and T cell co- WO9911791 stimulation can be determined using assays known in the art: Moore et al., 1999, Q9N568/TNR19_HUMAN Science, 285(5425): 260-3; Song H Y et al., 1997 Proc Natl Acad Sci USA 94(18): Tumor necrosis factor 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Immune Disorders, receptor superfamily Cancers member 19 (isoform 1) (SEQ ID NO: 324) TNF-alpha precursor Activities associated with apoptosis, NF-kB activation, and co- stimulation of immune GeneSeq Accession cells such as T and B cells. Apoptosis activity, NF-kB activation, and B and T cell co- P60074 EP205038 stimulation can be determined using assays known in the art: Moore et al., 1999, Science, 285(5425): 260-3; Song H Y et al., 1997 Proc Natl Acad Sci USA 94(18): 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Inflammatory disorders immunologic disorders, cancer Human TNF- alpha Activities associated with apoptosis, NF-kB activation, and co- stimulation of immune GeneSeq Accession cells such as T and B cells. Apoptosis activity, NF-kB activation, and B and T cell co- R62463 EP619372 stimulation can be determined using assays known in the art: Moore et al., 1999, P01375/TNFA_HUMAN Science, 285(5425): 260-3; Song H Y et al., 1997 Proc Natl Acad Sci USA 94(18): Tumor necrosis factor 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Inflammatory (SEQ ID NO: 325) disorders immunologic disorders, cancer Human TNF- alpha Activities associated with apoptosis, NF-kB activation, and co- stimulation of immune GeneSeq Accession cells such as T and B cells. Apoptosis activity, NF-kB activation, and B and T cell co- R42679 EP563714 stimulation can be determined using assays known in the art: Moore et al., 1999, P01375/TNFA_HUMAN Science, 285(5425): 260-3; Song H Y et al., 1997 Proc Natl Acad Sci USA 94(18): Tumor necrosis factor 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Inflammatory (SEQ ID NO: 325) disorders immunologic disorders, cancer Human TNF- beta (LT- Activities associated with apoptosis, NF-kB activation, and co- stimulation of immune alpha) GeneSeq cells such as T and B cells. Apoptosis activity, NF-kB activation, and B and T cell co- Accession B37799 stimulation can be determined using assays known in the art: Moore et al., 1999, WO0064479 Science, 285(5425): 260-3; Song H Y et al., 1997 Proc Natl Acad Sci USA 94(18): P01374/TNFB_HUMAN 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Inflammatory Lymphotoxin-alpha disorders immunologic disorders, cancer (SEQ ID NO: 326) LT-alpha GeneSeq Activities associated with apoptosis, NF-kB activation, and co- stimulation of immune Accession P70107 cells such as T and B cells. Apoptosis activity, NF-kB activation, and B and T cell co- EP250000 stimulation can be determined using assays known in the art: Moore et al., 1999, P01374/TNFB_HUMAN Science, 285(5425): 260-3; Song H Y et al., 1997 Proc Natl Acad Sci USA 94(18): Lymphotoxin-alpha 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Inflammatory (SEQ ID NO: 326) disorders immunologic disorders, cancer LT-beta GeneSeq Activities associated with apoptosis, NF-kB activation, and co- stimulation of immune Accession R56869 cells such as T and B cells. Apoptosis activity, NF-kB activation, and B and T cell co- WO9413808 stimulation can be determined using assays known in the art: Moore et al., 1999, Q06643/TNFC_HUMAN Science, 285(5425): 260-3; Song H Y et al., 1997 Proc Natl Acad Sci USA 94(18): Lymphotoxin-beta 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Inflammatory (isoform 1) disorders immunologic disorders, cancer (SEQ ID NO: 327) OPGL GeneSeq Activities associated with apoptosis, NF-kB activation, and co- stimulation of immune Accession W83195 cells such as T and B cells. Apoptosis activity, NF-kB activation, and B and T cell co- WO9846751 stimulation can be determined using assays known in the art: Moore et al., 1999, O14788/TNF11_HUMAN Science, 285(5425): 260-3; Song H Y et al., 1997 Proc Natl Acad Sci USA 94(18): Tumor necrosis factor 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Inflammatory ligand superfamily disorders immunologic disorders, cancer, loss of bone mass member 11 (isoform 1) (SEQ ID NO: 328) FasL GeneSeq Accession Activities associated with apoptosis, NF-kB activation, and co- stimulation of immune W98071 WO9903999 cells such as T and B cells. Apoptosis activity, NF-kB activation, and B and T cell co- P48023/TNFL6_HUMAN stimulation can be determined using assays known in the art: Moore et al., 1999, Tumor necrosis factor Science, 285(5425): 260-3; Song H Y et al., 1997 Proc Natl Acad Sci USA 94(18): ligand superfamily 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Inflammatory member 6 disorders immunologic disorders, cancer (isoform 1) (SEQ ID NO: 329) FasL GeneSeq Accession Activities associated with apoptosis, NF-kB activation, and co- stimulation of immune W95041 WO9903998 cells such as T and B cells. Apoptosis activity, NF-kB activation, and B and T cell co- P48023/TNFL6_HUMAN stimulation can be determined using assays known in the art: Moore et al., 1999, Tumor necrosis factor Science, 285(5425): 260-3; Song H Y et al., 1997 Proc Natl Acad Sci USA 94(18): ligand superfamily 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Inflammatory member 6 disorders immunologic disorders, cancer (isoform 1) (SEQ ID NO: 329) CD27L GeneSeq Activities associated with apoptosis, NF-kB activation, and co- stimulation of immune Accession R50121 cells such as T and B cells. Apoptosis activity, NF-kB activation, and B and T cell co- WO9405691 stimulation can be determined using assays known in the art: Moore et al., 1999, P32970/CD70_HUMAN Science, 285(5425): 260-3; Song H Y et al., 1997 Proc Natl Acad Sci USA 94(18): CD70 antigen 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Inflammatory (isoform 1) disorders immunologic disorders, cancer (SEQ ID NO: 330) CD30 ligand GeneSeq Activities associated with apoptosis, NF-kB activation, and co- stimulation of immune Accession R45007 cells such as T and B cells. Apoptosis activity, NF-kB activation, and B and T cell co- WO9324135 stimulation can be determined using assays known in the art: Moore et al., 1999, P32971/TNFL8_HUMAN Science, 285(5425): 260-3; Song H Y et al., 1997 Proc Natl Acad Sci USA 94(18): Tumor necrosis factor 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Inflammatory ligand superfamily disorders immunologic disorders, cancer member 8 (SEQ ID NO: 331) CD40L GeneSeq Activities associated with apoptosis, NF-kB activation, and co- stimulation of immune Accession R85486 cells such as T and B cells. Apoptosis activity, NF-kB activation, and B and T cell co- WO9529935 stimulation can be determined using assays known in the art: Moore et al., 1999, P29965/CD40L_HUMAN Science, 285(5425): 260-3; Song H Y et al., 1997 Proc Natl Acad Sci USA 94(18): CD40 ligand 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Inflammatory (SEQ ID NO: 332) disorders immunologic disorders, cancer 4-1BB ligand GeneSeq Activities associated with apoptosis, NF-kB activation, and co- stimulation of immune Accession W26657 U.S. cells such as T and B cells. Apoptosis activity, NF-kB activation, and B and T cell co- Pat. No. 5,674,704 stimulation can be determined using assays known in the art: Moore et al., 1999, P41273/TNFL9_HUMAN Science, 285(5425): 260-3; Song H Y et al., 1997 Proc Natl Acad Sci USA 94(18): Tumor necrosis factor 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Inflammatory ligand superfamily disorders immunologic disorders, cancer member 9 (SEQ ID NO: 333) FAS Ligand Inhibitory Activities associated with apoptosis, NF-kB activation, and co- stimulation of immune Protein (DcR3) GeneSeq cells such as T and B cells. Apoptosis activity, NF-kB activation, and B and T cell co- Accession B19335 stimulation can be determined using assays known in the art: Moore et al., 1999, WO0058465 Science, 285(5425): 260-3; Song H Y et al., 1997 Proc Natl Acad Sci USA 94(18): O95407/TNF6B_HUMAN 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Soluble DcR3 Tumor necrosis factor polypeptides may be useful for inhibiting apoptosis, NF-kB activation, and/or co- receptor superfamily stimulation of immune cells such as B and T cells. member 6B (SEQ ID NO: 334) OX40L GeneSeq Activities associated with apoptosis, NF-kB activation, and co- stimulation of immune Accession R79903 cells such as T and B cells. Apoptosis activity, NF-kB activation, and B and T cell co- WO9521915 stimulation can be determined using assays known in the art: Moore et al., 1999, P23510/TNFL4_HUMAN Science, 285(5425): 260-3; Song H Y et al., 1997 Proc Natl Acad Sci USA 94(18): Tumor necrosis factor 9792-6; Epsevik and Nissen-Meyer, 1986, J. Immunol. Methods. Inflammatory ligand superfamily disorders immunologic disorders, cancer member 4 (isoform 1) (SEQ ID NO: 335) Protease inhibitor peptides Peptides that inhibit the function/binding of HIV HIV protease activities are known in GeneSeq Accessions the art: HIV protease assays: EP0387231. One can modify the assay to look for R12435, R12436, R12437, inhibition using any of the disclosed protease inhibitor polypeptides. HIV, R12438, R12439, R12440, inflammatory disorders, immuno- logic disorders, cancer, viral infections and R1244 WO9106561 Retroviral protease Peptides that inhibit the function/binding of HIV HIV protease activities are known in inhibitors GeneSeq the art: HIV protease assays: EP0387231. One can modify the assay to look for Accessions R06660, inhibition using any of the disclosed protease inhibitor polypeptides. HIV, R06661, R06662, R06663, inflammatory disorders, immuno- logic disorders, cancer, viral infections R06664, R06665, R06666, R06667, R06668, R06669, R06670, R06671, R06672, R06673, R06674, R06675, and R06676 EP387231 HIV protease inhibiting Peptides that inhibit the function/binding of HIV HIV protease activities are known in peptides GeneSeq the art: HIV protease assays: EP0387231. One can modify the assay to look for Accessions R59293, inhibition using any of the disclosed protease inhibitor polypeptides. HIV, R59294, R59295, R59296, inflammatory disorders, immuno- logic disorders, cancer, viral infections R59297, R59298, R59299, R592300, R59301, R59302, R59301, R59302, R59303, R59304, R59305, R59306, R59307, R59308, R59309, R59310, R59311, R59312, R59313, R59314, R59315, R59316, R59317 R59318, R59319, R59320, R59321, R59322, R59323, R59324, R59325, R59326, R59327, R59328, R59329, R59330, R59331, R59332, R59333, R59334, R59335, R59336, R59337, R59338, R59339, R59340, R59341, R59342, R59343, R59344, R59345, R59346, R59347, R59348, R59349, and R59350 WO9301828 HIV-1 protease inhibitors Peptides that inhibit the function/binding of HIV HIV protease activities are known in GeneSeq Accessions the art: HIV protease assays: EP0387231. One can modify the assay to look for R86326, R86327, R86328, inhibition using any of the disclosed protease inhibitor polypeptides. HIV, R86329, R86330, R86331, inflammatory disorders, immuno- logic disorders, cancer, viral infections R86332, R86333, R86334, R86335, R86336, R86337, R86338, R86339, R86340, R86341, R86342, R86343, R86344, R86345, R86346, R86347, R86348, R86349, R86350, R86351, R86352, R86353, R86354, R86355, R86356, R86357, R86358, R86359, R86360, R86361, R86362, R86363, R86364, R86365, R86366, R86367, R86368, R86369, R86370, and R86371 DE4412174 HIV Inhibitor Peptide Peptides that inhibit the function/binding of HIV HIV protease activities are known in GeneSeq Accession the art: HIV protease assays: EP0387231. One can modify the assay to look for Y89687 WO9959615 inhibition using any of the disclosed protease inhibitor polypeptides. HIV, inflammatory disorders, immuno- logic disorders, cancer, viral infections HIV Inhibitor Peptide Peptides that inhibit the function/binding of HIV HIV protease activities are known in GenSeq Accession the art: HIV protease assays: EP0387231. One can modify the assay to look for Y31955 WO9948513 inhibition using any of the disclosed protease inhibitor polypeptides. HIV, inflammatory disorders, immuno- logic disorders, cancer, viral infections HIV Inhibitor Peptide Peptides that inhibit the function/binding of HIV HIV protease activities are known in Science 291, 884 (2001); the art: HIV protease assays: EP0387231. One can modify the assay to look for Published online 12 Jan. inhibition using any of the disclosed protease inhibitor polypeptides. HIV, 2001; 10.1126/science.1057453 inflammatory disorders, immuno- logic disorders, cancer, viral infections Human monocyte Chemokines are a family of related small, secreted proteins involved in biological chemoattractant factor processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. hMCP-3 GeneSeq Members of this family are involved in a similarly diverse range of pathologies Accession R73915 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The WO9509232 chemokines exert their effects by acting on a family of seven transmembrane G- P80098/CCL7_HUMAN protein coupled receptors. Over 40 human chemokines have been described, which C-C motif chemokine 7 bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 336) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, particularly useful for treating bacterial and/or viral menigitis Human monocyte Chemokines are a family of related small, secreted proteins involved in biological chemoattractant factor processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. hMCP-1 GeneSeq Members of this family are involved in a similarly diverse range of pathologies Accession R73914 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The WO9509232 chemokines exert their effects by acting on a family of seven transmembrane G- P13500/CCL2_HUMAN protein coupled receptors. Over 40 human chemokines have been described, which C-C motif chemokine 2 bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 337) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, particularly useful for treating bacterial and/or viral menigitis Human gro-beta Chemokines are a family of related small, secreted proteins involved in biological chemokine GeneSeq processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Accessions R66699 and Members of this family are involved in a similarly diverse range of pathologies W17671 WO9429341 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The P19875/CXCL2_HUMAN chemokines exert their effects by acting on a family of seven transmembrane G- C-X-C motif chemokine 2 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 338) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, inflammatory dis- orders, blood- related disorders, stem cell transplantation, cancer Human gro-gamma Chemokines are a family of related small, secreted proteins involved in biological chemokine GeneSeq processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Accessions R66700 and Members of this family are involved in a similarly diverse range of pathologies W17672 WO9429341 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The P19876/CXCL3_HUMAN chemokines exert their effects by acting on a family of seven transmembrane G- C-X-C motif chemokine 3 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 339) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, inflammatory dis- orders, blood- related disorders, stem cell transplantation, cancer Human gro-alpha Chemokines are a family of related small, secreted proteins involved in biological chemokine GeneSeq processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Accessions R66698 and Members of this family are involved in a similarly diverse range of pathologies W18024 WO9429341 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The P09341/GROA_HUMAN chemokines exert their effects by acting on a family of seven transmembrane G- Growth-regulated alpha protein coupled receptors. Over 40 human chemokines have been described, which protein bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 340) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, inflammatory disorders, blood- related disorders, stem cell transplantation, cancer Human eosinophil- Chemokines are a family of related small, secreted proteins involved in biological expressed chemokine processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. (EEC) GeneSeq Members of this family are involved in a similarly diverse range of pathologies Accession W05186 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The WO9632481 chemokines exert their effects by acting on a family of seven transmembrane G- SEQ ID NO: 2 of protein coupled receptors. Over 40 human chemokines have been described, which WO9632481 bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 401) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, particularly treatment of eosinophilia, inflammation, allergies, asthma, leukaemia and lymphoma Chemokine-like protein Chemokines are a family of related small, secreted proteins involved in biological PF4-414 Full-Length and processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Mature GeneSeq Members of this family are involved in a similarly diverse range of pathologies Accessions R92318 and including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The R99809 WO9613587 chemokines exert their effects by acting on a family of seven transmembrane G- FIG. 3C of WO9613587 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 402) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Cancer and blood- related disorders, particularly myelosuppression Chemokine-like protein IL- Chemokines are a family of related small, secreted proteins involved in biological 8M3 GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. R99812 WO9613587 Members of this family are involved in a similarly diverse range of pathologies including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The chemokines exert their effects by acting on a family of seven transmembrane G- protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ; and Holmes et al (1991) Science 253, 1278-80. Cancer and blood- related disorders, particularly myelosuppression Human interleukin-8 (IL-8) Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. R99814 WO9613587 Members of this family are involved in a similarly diverse range of pathologies P10145/IL8_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The Interleukin-8 chemokines exert their effects by acting on a family of seven transmembrane G- (isoform 1) protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 341) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ; and Holmes et al (1991) Science 253, 1278-80. Cancer and blood- related disorders, particularly myelosuppression Chemokine-like protein IL- Chemokines are a family of related small, secreted proteins involved in biological 8M1 Full-Length and processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Mature GeneSeq Members of this family are involved in a similarly diverse range of pathologies Accessions R99815 and including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The R99803 WO9613587 chemokines exert their effects by acting on a family of seven transmembrane G- FIG. 4B of WO9613587 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 403) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ; and Holmes et al (1991) Science 253, 1278-80. Cancer and blood- related disorders, particularly myelosuppression Chemokine-like protein IL- Chemokines are a family of related small, secreted proteins involved in biological 8M8 Full-Length and processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Mature GeneSeq Members of this family are involved in a similarly diverse range of pathologies Accessions R99816 and including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The R99805 WO9613587 chemokines exert their effects by acting on a family of seven transmembrane G- FIG. 4C of WO9613587 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 404) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ; and Holmes et al (1991) Science 253, 1278-80. Cancer and blood- related disorders, particularly myelosuppression Chemokine-like protein IL- Chemokines are a family of related small, secreted proteins involved in biological 8M8 Full-Length and processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Mature GeneSeq Members of this family are involved in a similarly diverse range of pathologies Accessions R99817 and including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The R99806 WO9613587 chemokines exert their effects by acting on a family of seven transmembrane G- FIG. 4C of WO9613587 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 404) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ; and Holmes et al (1991) Science 253, 1278-80. Cancer and blood- related disorders, particularly myelosuppression. Chemokine-like protein IL- Chemokines are a family of related small, secreted proteins involved in biological 8M8 Full-Length and processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Mature GeneSeq Members of this family are involved in a similarly diverse range of pathologies Accessions R99818 and including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The R99804 WO9613587 chemokines exert their effects by acting on a family of seven transmembrane G- FIG. 4C of WO9613587 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 404) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ; and Holmes et al (1991) Science 253, 1278-80. Cancer and blood- related disorders, particularly myelosuppression. Chemokine-like protein IL- Chemokines are a family of related small, secreted proteins involved in biological 8M8 Full-Length and processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Mature GeneSeq Members of this family are involved in a similarly diverse range of pathologies Accessions R99819 and including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The R99807 WO9613587 chemokines exert their effects by acting on a family of seven transmembrane G- FIG. 4C of WO9613587 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 404) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Cancer and blood- related disorders, particularly myelosuppression. Chemokine-like protein IL- Chemokines are a family of related small, secreted proteins involved in biological 8M8 Full-Length and processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Mature GeneSeq Members of this family are involved in a similarly diverse range of pathologies Accessions R99822 and including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The R9807 WO9613587 chemokines exert their effects by acting on a family of seven transmembrane G- FIG. 4C of WO9613587 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 404) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Cancer and blood- related disorders, particularly myelosuppression. Human foetal spleen expressed Chemokines are a family of related small, secreted proteins involved in biological chemo-kine, processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. FSEC GeneSeq Members of this family are involved in a similarly diverse range of pathologies Accession R98499 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The WO9622374 chemokines exert their effects by acting on a family of seven transmembrane G- SEQ ID NO: 2 of protein coupled receptors. Over 40 human chemokines have been described, which WO9622374 bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 405) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders Liver expressed Chemokines are a family of related small, secreted proteins involved in biological chemokine-1(LVEC-1) processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies R95689 WO9616979 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The SEQ ID NO: 2 of chemokines exert their effects by acting on a family of seven transmembrane G- WO9616979 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 406) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Inflammation of the liver Liver expressed Chemokines are a family of related small, secreted proteins involved in biological chemokine-2(LVEC-2) processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies R95690 WO9616979 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The SEQ ID NO: 4 of chemokines exert their effects by acting on a family of seven transmembrane G- WO9616979 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 407) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Inflammation of the liver Pituitary expressed Chemokines are a family of related small, secreted proteins involved in biological chemokine (PGEC) processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies R95691 WO9616979 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The SEQ ID NO: 6 of chemokines exert their effects by acting on a family of seven transmembrane G- WO9616979 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 408) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Inflammation, particularly of the liver Adenoid-expressed Chemokines are a family of related small, secreted proteins involved in biological chemokine (ADEC) processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies R97664 WO9617868 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The SEQ ID NO: 2 of chemokines exert their effects by acting on a family of seven transmembrane G- WO9617868 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 409) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Inflammation, angiogenesis, tumorigenesis, musculoskeletal disorders Human chemokine CC-2 Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. W38170 WO9741230 Members of this family are involved in a similarly diverse range of pathologies Q16663/CCL15_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The C-C motif chemokine 15 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 342) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, cell migration, proliferation, and differentiation disorders Human chemokine HCC-1 Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. W38171 WO9741230 Members of this family are involved in a similarly diverse range of pathologies Q16627/CCL14_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The C-C motif chemokine 14 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 343) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, cell migration, proliferation, and differentiation disorders Human chemokine CC- 3 Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. W38172 WO9741230 Members of this family are involved in a similarly diverse range of pathologies Q16627/CCL14_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The C-C motif chemokine 14 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 343) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, cell migration, proliferation, and differentiation Novel betachemokine Chemokines are a family of related small, secreted proteins involved in biological designated PTEC processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies W27271 WO9739126 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The SEQ ID NO: 2 of chemokines exert their effects by acting on a family of seven transmembrane G- WO9739126 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 410) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, vascular disorders, cancer Human CX3C 111 amino Chemokines are a family of related small, secreted proteins involved in biological acid chemokine GeneSeq processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Accession W23344 Members of this family are involved in a similarly diverse range of pathologies WO9727299 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The SEQ ID NO: 2 of chemokines exert their effects by acting on a family of seven transmembrane G- WO9727299 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 411) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, inflammatory diseases, abnormal proliferation, regeneration, degeneration, and atrophy Human CCF18 chemokine Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. W25942 WO9721812 Members of this family are involved in a similarly diverse range of pathologies SEQ ID NO: 4 of including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The WO9721812 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 412) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Abnormal physiology and development disorders, can also be used as an anti- viral agent Human beta- chemokine Chemokines are a family of related small, secreted proteins involved in biological H1305 (MCP-2) GeneSeq processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Accession W26655 Members of this family are involved in a similarly diverse range of pathologies WO9725427 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The P80075/CCL8_HUMAN chemokines exert their effects by acting on a family of seven transmembrane G- C-C motif chemokine 8 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 344) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Chemotaxis, blood- related disorders, viral infection, HIV, wound healing, cancer Human eosinocyte CC Chemokines are a family of related small, secreted proteins involved in biological type chemokine eotaxin processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies W14990 WO9712914 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The P51671/CCL11_HUMAN chemokines exert their effects by acting on a family of seven transmembrane G- Eotaxin protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 245) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Inflammatory and immune disorders Human thymus and Chemokines are a family of related small, secreted proteins involved in biological activation regulated processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. cytokine (TARC) GeneSeq Members of this family are involved in a similarly diverse range of pathologies Accession W14018 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The WO9711969 chemokines exert their effects by acting on a family of seven transmembrane G- Q92583/CCL17_HUMAN protein coupled receptors. Over 40 human chemokines have been described, which C-C motif chemokine 17 bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 261) using assays known in the art: Methods in molecular Biology, 2000, vol. 138: Chemokine Protocols, Edited by A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power Humana Press Inc., Totowa, NJ Inflammatory and immune disorders Human chemokine beta-8 Chemokines are a family of related small, secreted proteins involved in biological short forms GeneSeq processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Accession W16315 Members of this family are involved in a similarly diverse range of pathologies WO9712041 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The Wildtype chemokine beta- chemokines exert their effects by acting on a family of seven transmembrane G- 8 provided as: protein coupled receptors. Over 40 human chemokines have been described, which P55773|CCL23_HUMAN bind to ~17 receptors thus far identified. Chemokine activities can be determined C-C motif chemokine 23 using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: (SEQ ID NO: 459) Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Cancer, wound healing, immune disorders Microphage derived Chemokines are a family of related small, secreted proteins involved in biological chemokine, MDC processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies W20058 WO9640923 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The O00626/CCL22_HUMAN chemokines exert their effects by acting on a family of seven transmembrane G- C-C motif chemokine 22 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 345) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Inflammatory diseases, wound healin, angiogenesis Human chemokine ZSIG- Chemokines are a family of related small, secreted proteins involved in biological 35 GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. W30565 WO9844117 Members of this family are involved in a similarly diverse range of pathologies SEQ ID NO: 2 of WO including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The WO9844117 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 413) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Inflammatory and immune diseases Primate CC chemokine Chemokines are a family of related small, secreted proteins involved in biological “ILINCK” GeneSeq processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Accesssion W69990 Chemokine activities can be determined using assays known in the art: Methods in WO98328658 Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. SEQ ID NO: 4 from Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. WO9832858 Immune and inflammatory disorders, abnormal proliferation, regen- eration, (SEQ ID NO: 414) generation and atrophy disorders Primate CXC chemokine Chemokines are a family of related small, secreted proteins involved in biological “IBICK” GeneSeq processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Accession W69989 Members of this family are involved in a similarly diverse range of pathologies WO9832858 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The SEQ ID NO: 2 from chemokines exert their effects by acting on a family of seven transmembrane G- WO9832858 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 415) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune and inflammatory disorders, abnormal proliferation, regen- eration, generation and atrophy disorders Human CC-type Chemokines are a family of related small, secreted proteins involved in biological chemokine protein processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. designated SLC Members of this family are involved in a similarly diverse range of pathologies (secondary lymphoid including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The chemokine) GeneSeq chemokines exert their effects by acting on a family of seven transmembrane G- Accession W69163 protein coupled receptors. Over 40 human chemokines have been described, which WO9831809 bind to ~17 receptors thus far identified. Chemokine activities can be determined O00585/CCL21_HUMAN using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: C-C motif chemokine 21 Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. (SEQ ID NO: 346) Humana Press Inc., Totowa, NJ. Immune, inflammatory, and infectious disorders, cancer Human CC chemokine Chemokines are a family of related small, secreted proteins involved in biological ELC protein GeneSeq processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Accession W62542 Members of this family are involved in a similarly diverse range of pathologies WO9826071 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The Q99731/CCL19_HUMAN chemokines exert their effects by acting on a family of seven transmembrane G- C-C motif chemokine 19 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 249) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Cancer and infectious diseases, particularly herpes virus Human DVic-1 C-C Chemokines are a family of related small, secreted proteins involved in biological chemokine GeneSeq processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Accession W60649 Members of this family are involved in a similarly diverse range of pathologies WO9823750 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The SEQ ID NO: 2 of chemokines exert their effects by acting on a family of seven transmembrane G- WO9823750 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 416) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Abnormal prolifera- tion, regeneration, degeneration, and atrophy disorders, including cancer Human C-C chemokine Chemokines are a family of related small, secreted proteins involved in biological DGWCC GeneSeq processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Accession W60650 Members of this family are involved in a similarly diverse range of pathologies WO9823750 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The SEQ ID NO: 6 of chemokines exert their effects by acting on a family of seven transmembrane G- WO9823750 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 417) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, cell proliferation disorders, cancer Human STCP-1 GeneSeq Chemokines are a family of related small, secreted proteins involved in biological Accession W62783 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. WO9824907 Members of this family are involved in a similarly diverse range of pathologies O00626/CCL22_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The C-C motif chemokine 22 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 345) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, particularly T cell related disorders, viral infection, and inflammation, especially joint Exodus protein GeneSeq Chemokines are a family of related small, secreted proteins involved in biological Accession W61279 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. WO9821330 Members of this family are involved in a similarly diverse range of pathologies P78556/CCL20_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The C-C motif chemokine 20 chemokines exert their effects by acting on a family of seven transmembrane G- (isoform 1) protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 248) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune and inflammatory disorders, angiogenesis, cancer, and proliferation disorders, particularly myeloproliferative diseases Human Chr19kine protein Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Acession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. W50887 WO9814581 Members of this family are involved in a similarly diverse range of pathologies SEQ ID NO: 10 of including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The WO9814581 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 418) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Cancer and de- generative disorders Human T cell mixed Chemokines are a family of related small, secreted proteins involved in biological lymphocyte reaction processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. expressed chemokine Members of this family are involved in a similarly diverse range of pathologies (TMEC) GeneSeq including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The Accession W58703 U.S. chemokines exert their effects by acting on a family of seven transmembrane G- Pat. No. 5,780,268 protein coupled receptors. Over 40 human chemokines have been described, which SEQ ID NO: 2 of U.S. Pat. bind to ~17 receptors thus far identified. Chemokine activities can be determined No. 5,780,268 using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: (SEQ ID NO: 460) Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune, inflammatory, and infectious disorders, cancer Human 6CKine protein Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. W50885 W09814581 Members of this family are involved in a similarly diverse range of pathologies SEQ ID NO: 8 of including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The WO9814581 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 419) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Cancer and de- generative disorders human liver and activation Chemokines are a family of related small, secreted proteins involved in biological regulated chemokine processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. (LARC) GeneSeq Members of this family are involved in a similarly diverse range of pathologies Accession W57475 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The WO9817800 chemokines exert their effects by acting on a family of seven transmembrane G- P78556/CCL20_HUMAN protein coupled receptors. Over 40 human chemokines have been described, which C-C motif chemokine 20 bind to ~17 receptors thus far identified. Chemokine activities can be determined (isoform 1) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: (SEQ ID NO: 248) Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune, inflammatory, and infectious disorders, cancer RANTES peptide Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. W29538 WO9744462 Members of this family are involved in a similarly diverse range of pathologies Wildtype Rantes provided including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The herien as chemokines exert their effects by acting on a family of seven transmembrane G- P13501/CCL5_HUMAN protein coupled receptors. Over 40 human chemokines have been described, which C-C motif chemokine 5 bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 241) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Infectious diseases, particularly HIV RANTES 8-68 GeneSeq Chemokines are a family of related small, secreted proteins involved in biological Accession W29529 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. WO9744462 Members of this family are involved in a similarly diverse range of pathologies Wildtype Rantes provided including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The herein as chemokines exert their effects by acting on a family of seven transmembrane G- P13501/CCL5_HUMAN protein coupled receptors. Over 40 human chemokines have been described, which C-C motif chemokine 5 bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 241) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Infectious diseases, particularly HIV RANTES 9-68 GeneSeq Chemokines are a family of related small, secreted proteins involved in biological Accession W29528 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. WO9744462 Members of this family are involved in a similarly diverse range of pathologies Wildtype Rantes provided including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The herien as chemokines exert their effects by acting on a family of seven transmembrane G- P13501/CCL5_HUMAN protein coupled receptors. Over 40 human chemokines have been described, which C-C motif chemokine 5 bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 241) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Infectious diseases, particularly HIV Human chemokine protein Chemokines are a family of related small, secreted proteins involved in biological 331D5 GeneSeq processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Accession W59433 Members of this family are involved in a similarly diverse range of pathologies WO9811226 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The SEQ ID NO: 12 of chemokines exert their effects by acting on a family of seven transmembrane G- WO9811226 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 420) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Abnormal prolifera- tion, regeneration, degeneration, or atrophy, including cancer Human chemokine protein Chemokines are a family of related small, secreted proteins involved in biological 61164 GeneSeq processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Accession W59430 Members of this family are involved in a similarly diverse range of pathologies WO9811226 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The SEQ ID NO: 6 of chemokines exert their effects by acting on a family of seven transmembrane G- WO9811226 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 421) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Abnormal prolifera- tion, regeneration, degeneration, or atrophy, including cancer Chemokine MCP-4 Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. W56690 WO9809171 Members of this family are involved in a similarly diverse range of pathologies Q99616/CCL13_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The C-C motif chemokine 13 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 347) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune, Inflammatory, and infectious diseases Human stromal cell- Chemokines are a family of related small, secreted proteins involved in biological derived chemokine, SDF-1 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies W50766 FR2751658 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The P48061/SDF1_HUMAN chemokines exert their effects by acting on a family of seven transmembrane G- Stromal cell-derived factor 1 protein coupled receptors. Over 40 human chemokines have been described, which (isoform beta) bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 260) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. HIV infections Thymus expressed Chemokines are a family of related small, secreted proteins involved in biological chemokine (TECK) processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies W44397 WO9801557 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The O15444/CCL25_HUMAN chemokines exert their effects by acting on a family of seven transmembrane G- C-C motif chemokine 25 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 348) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune and inflammatory disorders Human chemokine MIP- Chemokines are a family of related small, secreted proteins involved in biological 3alpha GeneSeq processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Accession W44398 Members of this family are involved in a similarly diverse range of pathologies WO9801557 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The P78556/CCL20_HUMAN chemokines exert their effects by acting on a family of seven transmembrane G- C-C motif chemokine 20 protein coupled receptors. Over 40 human chemokines have been described, which (isoform 1) bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 248) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune and inflammatory disorders Human chemokine MIP- Chemokines are a family of related small, secreted proteins involved in biological 3beta GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. W44399 WO9801557 Members of this family are involved in a similarly diverse range of pathologies Q99731/CCL19_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The C-C motif chemokine 19 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 249) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune and inflammatory disorders Human monocyte Chemokines are a family of related small, secreted proteins involved in biological chemotactic proprotein processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. (MCPP) sequence Members of this family are involved in a similarly diverse range of pathologies GeneSeq Accession including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The W42072 WO9802459 chemokines exert their effects by acting on a family of seven transmembrane G- SEQ ID NO: 1 of protein coupled receptors. Over 40 human chemokines have been described, which WO9802459 bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 456) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, respiratory disorders, cancer Macrophage- derived Chemokines are a family of related small, secreted proteins involved in biological chemokine (MDC) processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeq Accessions Members of this family are involved in a similarly diverse range of pathologies W40811 and Y24414 U.S. including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The Pat. No. 5,688,927/U.S. chemokines exert their effects by acting on a family of seven transmembrane G- Pat. No. 5,932,703 protein coupled receptors. Over 40 human chemokines have been described, which O00626/CCL22_HUMAN bind to ~17 receptors thus far identified. Chemokine activities can be determined C-C motif chemokine 22 using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: (SEQ ID NO: 345) Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune, and inflammatory disorders, cancer Macrophage derived Chemokines are a family of related small, secreted proteins involved in biological chemokine analogue processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. MDC-eyfy GeneSeq Members of this family are involved in a similarly diverse range of pathologies Accession Y24416 U.S. including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The Pat. No. 5,932,703 (“eyfy” chemokines exert their effects by acting on a family of seven transmembrane G- disclosed as SEQ ID NO: protein coupled receptors. Over 40 human chemokines have been described, which 546) bind to ~17 receptors thus far identified. Chemokine activities can be determined Wildtype MDC is SEQ ID using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: NO: 2 of 5,932,703 Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. (SEQ ID NO: 422) Humana Press Inc., Totowa, NJ. Immune and inflammatory disorders Macrophage derived Chemokines are a family of related small, secreted proteins involved in biological chemokine analogue MDC processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. (n + 1) GeneSeq Members of this family are involved in a similarly diverse range of pathologies Accession Y24413 U.S. including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The Pat. No. 5,932,703 chemokines exert their effects by acting on a family of seven transmembrane G- protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune and inflammatory disorders Macrophage derived Chemokines are a family of related small, secreted proteins involved in biological chemokine analogue processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. MDC-yl GeneSeq Members of this family are involved in a similarly diverse range of pathologies Accession Y24415 U.S. including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The Pat. No. 5,932,703 chemokines exert their effects by acting on a family of seven transmembrane G- protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune and inflammatory disorders Human type CC Chemokines are a family of related small, secreted proteins involved in biological chemokine eotaxin 3 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. protein sequence Members of this family are involved in a similarly diverse range of pathologies GeneSeq Accession including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The Y43178 JP11243960 chemokines exert their effects by acting on a family of seven transmembrane G- Q9Y258/CCL26_HUMAN protein coupled receptors. Over 40 human chemokines have been described, which C-C motif chemokine 26 bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 349) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Allergic diseases and HIV infection Human MCP-3 and human Chemokines are a family of related small, secreted proteins involved in biological Muc-1 core epitope (VNT) processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. fusion protein GeneSeq Members of this family are involved in a similarly diverse range of pathologies Acession Y29893 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The WO9946392 chemokines exert their effects by acting on a family of seven transmembrane G- Wildtype MCP-3 has the protein coupled receptors. Over 40 human chemokines have been described, which sequence: bind to ~17 receptors thus far identified. Chemokine activities can be determined P80098/CCL7_HUMAN using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: C-C motif chemokine 7 Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. (SEQ ID NO: 336) Humana Press Inc., Totowa, NJ. Cancer and immune disorders, particularly HIV Wildtype Muc-1 has the infection sequence: P15941|MUC1_HUMAN Mucin-1 (isoform 1) (SEQ ID NO: 461) Human IP-10 and human Chemokines are a family of related small, secreted proteins involved in biological Muc-1 core epitope (VNT) processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. fusion protein GeneSeq Members of this family are involved in a similarly diverse range of pathologies Accession Y29894 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The WO9946392 chemokines exert their effects by acting on a family of seven transmembrane G- Wildtype IP10 has the protein coupled receptors. Over 40 human chemokines have been described, which sequence: bind to ~17 receptors thus far identified. Chemokine activities can be determined P02778/CXL10_HUMAN using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: C-X-C motif chemokine 10 Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. (SEQ ID NO: 242) Humana Press Inc., Totowa, NJ. Cancer and immune disorders, particularly HIV Wildtype Muc-1 has the infection sequence: P15941|MUC1_HUMAN Mucin-1 (isoform 1) (SEQ ID NO: 461) Human IP-10 and HIV-1 Chemokines are a family of related small, secreted proteins involved in biological gp 120 hyper- variable processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. region fusion protein Members of this family are involved in a similarly diverse range of pathologies GeneSeq Accession including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The Y29897 W09946392 chemokines exert their effects by acting on a family of seven transmembrane G- Wildtype IP10 has the protein coupled receptors. Over 40 human chemokines have been described, which sequence: bind to ~17 receptors thus far identified. Chemokine activities can be determined P02778/CXL10_HUMAN using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: C-X-C motif chemokine 10 Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. (SEQ ID NO: 242) Humana Press Inc., Totowa, NJ. Cancer and immune disorders, particularly HIV Wildtype gp120 has the infection sequence: P03378|32-509 (cleaved product of gp160) (SEQ ID NO: 462) Human mammary Chemokines are a family of related small, secreted proteins involved in biological associated chemokine processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. (MACK) protein Full- Members of this family are involved in a similarly diverse range of pathologies Length and Mature including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The GeneSeq Accessions chemokines exert their effects by acting on a family of seven transmembrane G- Y29092 and Y29093 protein coupled receptors. Over 40 human chemokines have been described, which WO9936540 bind to ~17 receptors thus far identified. Chemokine activities can be determined Full-length: SEQ ID NO: 1 using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: of WO9936540 Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. (SEQ ID NO: 423) Humana Press Inc., Totowa, NJ. Breast disease, including cancer Mature Form: SEQ ID NO: 2 of WO9936540 (SEQ ID NO: 424) Tim-1 protein GeneSeq Chemokines are a family of related small, secreted proteins involved in biological Accession Y28290 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. WO9933990 Members of this family are involved in a similarly diverse range of pathologies SEQ ID NO: 2 of including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The WO9933990 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 350) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Inflammation due to stimuli such as heart attacks and stroke, infection, physical trauma, UV or ionizing radiation, burns, frostbite or corrosive chemicals Human Lkn-1 Full-Length Chemokines are a family of related small, secreted proteins involved in biological and Mature protein processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeq Accessions Members of this family are involved in a similarly diverse range of pathologies Y17280, Y17274, Y17281, including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The and Y17275 WO9928473 chemokines exert their effects by acting on a family of seven transmembrane G- and WO9928472 protein coupled receptors. Over 40 human chemokines have been described, which Q16663/CCL15_HUMAN bind to ~17 receptors thus far identified. Chemokine activities can be determined C-C motif chemokine 15 using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: (SEQ ID NO: 342) Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. HIV infection and cancer, particularly leukemia N-terminal modified Chemokines are a family of related small, secreted proteins involved in biological chemokine met- hSDF-1 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. alpha GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies Y05818 WO9920759 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The SEQ ID NO: 10 of chemokines exert their effects by acting on a family of seven transmembrane G- WO9920759 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 425) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Inhibit or stimulate angiogenesis, inhibit the binding of HIV N-terminal modified Chemokines are a family of related small, secreted proteins involved in biological chemokine met- hSDF-1 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. beta GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies Y05819 WO9920759 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The SEQ ID NO: 11 of chemokines exert their effects by acting on a family of seven transmembrane G- WO9920759 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 426) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Inhibit or stimulate angiogenesis, inhibit the binding of HIV, antiinflammatory; immunosuppressant N-terminal modified Chemokines are a family of related small, secreted proteins involved in biological chemokine processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GroHEK/hSDF- 1alpha Members of this family are involved in a similarly diverse range of pathologies GeneSeq Accession including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The Y05820 WO9920759 chemokines exert their effects by acting on a family of seven transmembrane G- SEQ ID NO: 12 of protein coupled receptors. Over 40 human chemokines have been described, which WO9920759 bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 427) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Inhibit or stimulate angiogenesis, inhibit the binding of HIV, antiinflammatory; immunosuppressant N-terminal modified Chemokines are a family of related small, secreted proteins involved in biological chemokine processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GroHEK/hSDF- 1beta. Members of this family are involved in a similarly diverse range of pathologies GeneSeq Accession including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The Y05821 WO9920759 chemokines exert their effects by acting on a family of seven transmembrane G- SEQ ID NO: 13 of protein coupled receptors. Over 40 human chemokines have been described, which WO9920759 bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 428) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Inhibit or stimulate angiogenesis, inhibit the binding of HIV, antiinflammatory; immunosuppressant Chemokine Eotaxin Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Y14230 WO9912968 Members of this family are involved in a similarly diverse range of pathologies P51671/CCL11_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The Eotaxin chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 245) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Increase or enhance an inflammatory response, an immune response orhaematopoietic cell-associated activity; treat a vascular indication; Cancer; enhance wound healing, to prevent or treat asthma, organ transplant rejction, rheumatoid arthritis or allergy Chemokine hMCP1a Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Y14225 WO9912968 Members of this family are involved in a similarly diverse range of pathologies including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The chemokines exert their effects by acting on a family of seven transmembrane G- protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, Vascular disorders, Wound healing, cancer, prevent organ transplant rejection, Increase or enhance an inflammatory response, Chemokine hMCP1b Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Y14226 WO9912968 Members of this family are involved in a similarly diverse range of pathologies including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The chemokines exert their effects by acting on a family of seven transmembrane G- protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, Vascular disorders, Wound healing, cancer, prevent organ transplant rejection, Increase or enhance an inflammatory response, Chemokine hSDF1b Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Y14228 WO9912968 Members of this family are involved in a similarly diverse range of pathologies P48061/SDF1_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The Stromal cell-derived factor 1 chemokines exert their effects by acting on a family of seven transmembrane G- (isoform beta) protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 260) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, Vascular disorders, Wound healing, cancer, prevent organ transplant rejection, Increase or enhance an inflammatory response, Chemokine hIL-8 Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Y14229 WO9912968 Members of this family are involved in a similarly diverse range of pathologies P10145/IL8_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The Interleukin-8 chemokines exert their effects by acting on a family of seven transmembrane G- (isoform 1) protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 341) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ; and Holmes et al (1991) Science 253, 1278-80. Immune disorders, Vascular disorders, Wound healing, cancer, prevent organ transplant rejection, Increase or enhance an inflammatory response, Chemokine hMCP1 Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Y14222 WO9912968 Members of this family are involved in a similarly diverse range of pathologies P13500/CCL2_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The C-C motif chemokine 2 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 337) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, Vascular disorders, Wound healing, cancer, prevent organ transplant rejection, Increase or enhance an inflammatory response, Chemokine hMCP2 Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Y14223 WO9912968 Members of this family are involved in a similarly diverse range of pathologies P80075/CCL8_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The C-C motif chemokine 8 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 344) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, Vascular disorders, Wound healing, cancer, prevent organ transplant rejection, Increase or enhance an inflammatory response, Chemokine hMCP3 Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Y14224 WO9912968 Members of this family are involved in a similarly diverse range of pathologies P80098/CCL7_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The C-C motif chemokine 7 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 336) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, Vascular disorders, Wound healing, cancer, prevent organ transplant rejection, Increase or enhance an inflammatory response, C-C chemokine, MCP2 Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Y05300 EP905240 Members of this family are involved in a similarly diverse range of pathologies P80075/CCL8_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The C-C motif chemokine 8 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 344) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Inflammatory, Immune and infectious diseases; pulmonary diseases and skin disorders; tumours, and angiogenesis-and haematopoiesis- related diseases Wild type monocyte Chemokines are a family of related small, secreted proteins involved in biological chemotactic protein 2 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies Y07233 EP906954 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The P80075/CCL8_HUMAN chemokines exert their effects by acting on a family of seven transmembrane G- C-C motif chemokine 8 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 344) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Inflammatory, Immune and infectious diseases; pulmonary diseases and skin disorders; tumours, and angiogenesis-and haematopoiesis- related diseases Truncated monocyte Chemokines are a family of related small, secreted proteins involved in biological chemotactic protein 2 (6- processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. 76) GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies Y07234 EP906954 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The FIG. 1 of EP905241 and chemokines exert their effects by acting on a family of seven transmembrane G- EP906954 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 429) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power, Humana Press Inc., Totowa, NJ Inflammatory, Immune and infectious diseases; pulmonary diseases and skin disorders; tumours, and angiogenesis-and haematopoiesis- related diseases Truncated RANTES Chemokines are a family of related small, secreted proteins involved in biological protein (3-68) GeneSeq processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Accessions Y07236 and Members of this family are involved in a similarly diverse range of pathologies Y07232 EP905241; including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The EP906954 chemokines exert their effects by acting on a family of seven transmembrane G- FIG. 1 of EP906954 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 430) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power, Humana Press Inc., Totowa, NJ Inflammatory, immune and infectious diseases; pulmonary diseases and skin disorders; tumours, and angiogenesis-and haematopoiesis- related diseases Wild type monocyte Chemokines are a family of related small, secreted proteins involved in biological chemotactic protein 2 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies Y07237 EP905241 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The P80075/CCL8_HUMAN chemokines exert their effects by acting on a family of seven transmembrane G- C-C motif chemokine 8 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 344) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power, Humana Press Inc., Totowa, NJ Inflammatory, immune and infectious diseases; pulmonary diseases and skin disorders; tumours, and angiogenesis-and haematopoiesis- related diseases Truncated monocyte Chemokines are a family of related small, secreted proteins involved in biological chemotactic protein 2 (6-76) processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies Y07238 EP905241 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The FIG. 1 of EP905241 and chemokines exert their effects by acting on a family of seven transmembrane G- EP906954 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 429) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power, Humana Press Inc., Totowa, NJ Inflammatory, immune and infectious diseases; pulmonary diseases and skin disorders; tumours, and angiogenesis-and haematopoiesis- related diseases A partial CXCR4B protein Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. W97363 EP897980 Members of this family are involved in a similarly diverse range of pathologies SEQ ID NO: 2 of including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The EP897980 chemokines exert their effects by acting on a family of seven transmembrane G- (sEQ ID NO: 431) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power, Humana Press Inc., Totowa, NJ Soluble CXCR4B receptor polypep- tides may be useful for inhibiting chemokine activities and viral infection. Interferon gamma- Chemokines are a family of related small, secreted proteins involved in biological inducible protein (IP-10) processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies W96709 U.S. Pat. No. including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The 5,871,723 chemokines exert their effects by acting on a family of seven transmembrane G- P02778/CXL10_HUMAN protein coupled receptors. Over 40 human chemokines have been described, which C-X-C motif chemokine 10 bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 242) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power, Humana Press Inc., Totowa, NJ Angiogenesis, Cancer, Inflammatory and Immune disorders, Cardio- Vascular disorders, Musco-skeletal disorders A monokine induced by Chemokines are a family of related small, secreted proteins involved in biological gamma- interferon (MIG) processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies W96710 U.S. Pat. No. including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The 5,871,723 chemokines exert their effects by acting on a family of seven transmembrane G- Q07325/CXCL9_HUMAN protein coupled receptors. Over 40 human chemokines have been described, which C-X-C motif chemokine 9 bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 351) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power, Humana Press Inc., Totowa, NJ Angiogenesis, Cancer, Inflammatory and Immune disorders, Cardio- Vascular disorders, Musco-skeletal disorders Interleukin-8 (IL-8) protein. Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. W96711 U.S. Pat. No. Members of this family are involved in a similarly diverse range of pathologies 5,871,723 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The P10145/IL8_HUMAN chemokines exert their effects by acting on a family of seven transmembrane G- Interleukin-8 protein coupled receptors. Over 40 human chemokines have been described, which (isoform 1) bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 341) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ; and Holmes et al (1991) Science 253, 1278-80. Angiogenesis, Cancer, Inflammatory and Immune disorders, Cardio- Vascular disorders, Musco-skeletal disorders Epithelial neutrophil Chemokines are a family of related small, secreted proteins involved in biological activating protein-78 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. (ENA-78) GeneSeq Members of this family are involved in a similarly diverse range of pathologies Accession W96712 U.S. including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The Pat. No. 5,871,723 chemokines exert their effects by acting on a family of seven transmembrane G- P42830/CXCL5_HUMAN protein coupled receptors. Over 40 human chemokines have been described, which C-X-C motif chemokine 5 bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 352) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power, Humana Press Inc., Totowa, NJ Angiogenesis, Cancer, Inflammatory and Immune disorders, Cardio- Vascular disorders, Musco-skeletal disorders Growth related oncogene- Chemokines are a family of related small, secreted proteins involved in biological alpha (GRO-alpha). processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies W96713 U.S. Pat. No. including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The 5,871,723 chemokines exert their effects by acting on a family of seven transmembrane G- P09341/GROA_HUMAN protein coupled receptors. Over 40 human chemokines have been described, which Growth-regulated alpha bind to ~17 receptors thus far identified. Chemokine activities can be determined protein using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: (SEQ ID NO: 340) Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power, Humana Press Inc., Totowa, NJ Angiogenesis, Cancer, Inflammatory and Immune disorders, Cardio- Vascular disorders, Musco-skeletal disorders Growth related oncogene- Chemokines are a family of related small, secreted proteins involved in biological beta (GRO-beta). processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies W96714 U.S. Pat. No. including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The 5,871,723 chemokines exert their effects by acting on a family of seven transmembrane G- P19875/CXCL2_HUMAN protein coupled receptors. Over 40 human chemokines have been described, which C-X-C motif chemokine 2 bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 338) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Angiogenesis, Cancer, Inflammatory and Immune disorders, Cardio- Vascular disorders, Musco-skeletal disorders Growth related oncogene- Chemokines are a family of related small, secreted proteins involved in biological gamma (GRO-gamma) processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies W96715 U.S. Pat. No. including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The 5,871,723 chemokines exert their effects by acting on a family of seven transmembrane G- P19876/CXCL3_HUMAN protein coupled receptors. Over 40 human chemokines have been described, which C-X-C motif chemokine 3 bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 339) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Angiogenesis, Cancer, Inflammatory and Immune disorders, Cardio- Vascular disorders, Musco-skeletal disorders A platelet basic protein Chemokines are a family of related small, secreted proteins involved in biological (PBP) GeneSeq processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Accession W96716 U.S. Members of this family are involved in a similarly diverse range of pathologies Pat. No. 5,871,723 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The P02775/CXCL7_HUMAN chemokines exert their effects by acting on a family of seven transmembrane G- Platelet basic protein protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 353) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Angiogenesis, Cancer, Inflammatory and Immune disorders, Cardio- Vascular disorders, Musco-skeletal disorders Connective tissue Chemokines are a family of related small, secreted proteins involved in biological activating protein-III processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. (CTAP-III) GeneSeqAccession Members of this family are involved in a similarly diverse range of pathologies S96717 U.S. Pat. including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The No. 5,871,723 chemokines exert their effects by acting on a family of seven transmembrane G- SEQ ID NO: 9 of U.S. protein coupled receptors. Over 40 human chemokines have been described, which Pat. No. 5,871,723 bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 354) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Angiogenesis, Cancer, Inflammatory and Immune disorders, Cardio- Vascular disorders, Musco-skeletal disorders Beta-thrombo- globulin Chemokines are a family of related small, secreted proteins involved in biological protein (beta-TG) processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies W96718 U.S. Pat. No. including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The 5,871,723 chemokines exert their effects by acting on a family of seven transmembrane G- SEQ ID NO: 10 of U.S. protein coupled receptors. Over 40 human chemokines have been described, which Pat. No. 5,871,723 bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 355) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Angiogenesis, Cancer, Inflammatory and Immune disorders, Cardio- Vascular disorders, Musco-skeletal disorders Neutrophil activating Chemokines are a family of related small, secreted proteins involved in biological peptide-2 (NAP-2) processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies W96719 U.S. Pat. No. including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The 5,871,723 chemokines exert their effects by acting on a family of seven transmembrane G- SEQ ID NO: 11 of U.S. protein coupled receptors. Over 40 human chemokines have been described, which Pat. No. 5,871,723 bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 356) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Angiogenesis, Cancer, Inflammatory and Immune disorders, Cardio- Vascular disorders, Musco-skeletal disorders Granulocyte chemotactic Chemokines are a family of related small, secreted proteins involved in biological protein-2 (GCP-2) processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies W96720 U.S. Pat. No. including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The 5,871,723 chemokines exert their effects by acting on a family of seven transmembrane G- P80162/CXCL6_HUMAN protein coupled receptors. Over 40 human chemokines have been described, which C-X-C motif chemokine 6 bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 357) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Angiogenesis, Cancer, Inflammatory and Immune disorders, Cardio- Vascular disorders, Musco-skeletal disorders Human chemokine MIG- Chemokines are a family of related small, secreted proteins involved in biological beta protein GeneSeq processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Accession W90124 Members of this family are involved in a similarly diverse range of pathologies EP887409 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The (SEQ ID NO: 463) chemokines exert their effects by acting on a family of seven transmembrane G- protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, viral, parasitic, fungal or bacterial infections, Cancer; autoimmune diseases or transplant rejection Human ZCHEMO-8 Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. W82716 WO9854326 Members of this family are involved in a similarly diverse range of pathologies SEQ ID NO: 2 of including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The WO9854326 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 432) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, cancer, myelopoietic disorders, autoimmune disorders and immunodeficiencies, Inflammatory and infectious diseases, Vascular disorders, wound healing Human Act-2 protein Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. W82717 WO9854326 Members of this family are involved in a similarly diverse range of pathologies P13236/CCL4_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The C-C motif chemokine 4 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 358) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, cancer, myelopoietic disorders, autoimmune disorders and immunodeficiencies, Inflammatory and infectious diseases, Vascular disorders, wound healing Human SISD protein Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Acession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. W82720 WO9854326 Members of this family are involved in a similarly diverse range of pathologies P13501/CCL5_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The C-C motif chemokine 5 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 241) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, cancer, myelopoietic disorders, autoimmune disorders and immunodeficiencies, Inflammatory and infectious diseases, Vascular disorders, wound healing Human MI10 protein Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. W82721 WO9854326 Members of this family are involved in a similarly diverse range of pathologies SEQ ID NO: 37 of including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The WO9854326 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 433) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, cancer, myelopoietic disorders, autoimmune disorders and immunodeficiencies, Inflammatory and infectious diseases, Vascular disorders, wound healing Human MI1A protein Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. W82722 W09854326 Members of this family are involved in a similarly diverse range of pathologies SEQ ID NO: 38 of including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The WO9854326 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 434) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, cancer, myelopoietic disorders, autoimmune disorders and immunodeficiencies, Inflammatory and infectious diseases, Vascular disorders, wound healing Human CCC3 protein Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. W82723 WO9854326 Members of this family are involved in a similarly diverse range of pathologies SEQ ID NO: 39 of including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The WO9854326 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 435) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune disorders, cancer, myelopoietic disorders, autoimmune disorders and immunodeficiencies, Inflammatory and infectious diseases, Vascular disorders, wound healing A human L105 chemokine Chemokines are a family of related small, secreted proteins involved in biological designated huL105_3. processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies W87588 WO9856818 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The SEQ ID NO: 2 of chemokines exert their effects by acting on a family of seven transmembrane G- WO9856818 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 436) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Cancer, wound healing A human L105 chemokine Chemokines are a family of related small, secreted proteins involved in biological designated huL105_7. processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies W87589 WO9856818 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The SEQ ID NO: 4 of chemokines exert their effects by acting on a family of seven transmembrane G- WO9856818 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 437) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Cancer, wound healing Human mature gro-alpha Chemokines are a family of related small, secreted proteins involved in biological polypeptide used to treat processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. sepsis GeneSeq Members of this family are involved in a similarly diverse range of pathologies Accession W81498 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The WO9848828 chemokines exert their effects by acting on a family of seven transmembrane G- P09341/GROA_HUMAN protein coupled receptors. Over 40 human chemokines have been described, which Growth-regulated alpha bind to ~17 receptors thus far identified. Chemokine activities can be determined protein using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: (SEQ ID NO: 340) Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Infectious diseases, sepsis Human mature gro- Chemokines are a family of related small, secreted proteins involved in biological gamma polypeptide used processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. to treat sepsis GeneSeq Members of this family are involved in a similarly diverse range of pathologies Accession W81500 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The WO9848828 chemokines exert their effects by acting on a family of seven transmembrane G- P19876/CXCL3_HUMAN protein coupled receptors. Over 40 human chemokines have been described, which C-X-C motif chemokine 3 bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 339) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Infectious diseases, sepsis Human thymus expressed Chemokines are a family of related small, secreted proteins involved in biological chemokine TECK and processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. TECK variant GeneSeq Members of this family are involved in a similarly diverse range of pathologies Accessions B19607 and including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The B19608 WO0053635 chemokines exert their effects by acting on a family of seven transmembrane G- Wildtype TECK provided protein coupled receptors. Over 40 human chemokines have been described, which as: bind to ~17 receptors thus far identified. Chemokine activities can be determined O15444/CCL25_HUMAN using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: C-C motif chemokine 25 Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. (SEQ ID NO: 348) Humana Press Inc., Totowa, NJ. Inflammatory disorders, cancer, Immune and vascular disorders Human chemokine Chemokines are a family of related small, secreted proteins involved in biological SDF1alpha GeneSeq processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Accession B15791 Members of this family are involved in a similarly diverse range of pathologies WO0042071 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The P48061-2/SDF1_HUMAN chemokines exert their effects by acting on a family of seven transmembrane G- Isoform Alpha of Stromal protein coupled receptors. Over 40 human chemokines have been described, which cell-derived factor 1 bind to ~17 receptors thus far identified. Chemokine activities can be determined (isoform alpha) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: (SEQ ID NO: 259) Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Autoimmune disorders, Immune, Vascular and Inflammatory disorders Human chemokine GRO- Chemokines are a family of related small, secreted proteins involved in biological alpha GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. B15793 WO0042071 Members of this family are involved in a similarly diverse range of pathologies P09341/GROA_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The Growth-regulated alpha chemokines exert their effects by acting on a family of seven transmembrane G- protein protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 340) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Autoimmune disorders, Immune, Vascular and Inflammatory disorders Human chemokine eotaxin Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. B15794 WO0042071 Members of this family are involved in a similarly diverse range of pathologies P51671/CCL11_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The Eotaxin chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 245) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Autoimmune disorders, Immune, Vascular and Inflammatory disorders Human chemokine MIG Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. B15803 WO0042071 Members of this family are involved in a similarly diverse range of pathologies Q07325/CXCL9_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The C-X-C motif chemokine 9 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 351) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Autoimmune disorders, Immune, Vascular and Inflammatory disorders Human chemokine PF4 Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. B15804 WO0042071 Members of this family are involved in a similarly diverse range of pathologies P02776/PLF4_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The Platelet factor 4 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 359) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Autoimmune disorders, Immune, Vascular and Inflammatory disorders Human chemokine I-309 Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. B15805 WO0042071 Members of this family are involved in a similarly diverse range of pathologies P22362/CCL1_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The C-C motif chemokine 1 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 360) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Autoimmune disorders, Immune, Vascular and Inflammatory disorders Human chemokine HCC-1 Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. B15806 WO0042071 Members of this family are involved in a similarly diverse range of pathologies Q16627/CCL14_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The C-C motif chemokine 14 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 361) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Autoimmune disorders, Immune, Vascular and Inflammatory disorders Human chemokine C10 Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. B15807 WO0042071 Members of this family are involved in a similarly diverse range of pathologies SEQ ID NO: 49 of including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The WO0042071 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 438) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Autoimmune disorders, Immune, Vascular and Inflammatory disorders Human chemokine CCR-2 Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. B15808 WO0042071 Members of this family are involved in a similarly diverse range of pathologies P41597/CCR2_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The C-C chemokine receptor chemokines exert their effects by acting on a family of seven transmembrane G- type 2 protein coupled receptors. Over 40 human chemokines have been described, which (isoform A) bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 362) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Autoimmune disorders, Immune, Vascular and Inflammatory disorders Human chemokine ENA- Chemokines are a family of related small, secreted proteins involved in biological 78 GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. B15809 WO0042071 Members of this family are involved in a similarly diverse range of pathologies P42830/CXCL5_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The C-X-C motif chemokine 5 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 352) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Autoimmune disorders, Immune, Vascular and Inflammatory disorders Human chemokine Chemokines are a family of related small, secreted proteins involved in biological GRObeta GeneSeq processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Accession B15810 Members of this family are involved in a similarly diverse range of pathologies WO0042071 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The P19875/CXCL2_HUMAN chemokines exert their effects by acting on a family of seven transmembrane G- C-X-C motif chemokine 2 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 338) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Autoimmune disorders, Immune, Vascular and Inflammatory disorders Human chemokine IP-10 Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. B15811 WO0042071 Members of this family are involved in a similarly diverse range of pathologies P02778/CXL10_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The C-X-C motif chemokine 10 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 242) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Autoimmune disorders, Immune, Vascular and Inflammatory disorders Human chemokine Chemokines are a family of related small, secreted proteins involved in biological SDF1beta GeneSeq processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Accession B15812 Members of this family are involved in a similarly diverse range of pathologies WO0042071 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The P48061/SDF1_HUMAN chemokines exert their effects by acting on a family of seven transmembrane G- Stromal cell-derived factor 1 protein coupled receptors. Over 40 human chemokines have been described, which (isoform beta) bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 260) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Autoimmune disorders, Immune, Vascular and Inflammatory disorders Human chemokine GRO Chemokines are a family of related small, secreted proteins involved in biological alpha GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. B15813 WO0042071 Members of this family are involved in a similarly diverse range of pathologies P09341/GROA_HUMAN including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The Growth-regulated alpha chemokines exert their effects by acting on a family of seven transmembrane G- protein protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 340) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Autoimmune disorders, Immune, Vascular and Inflammatory disorders Human chemokine Chemokines are a family of related small, secreted proteins involved in biological MIP1beta GeneSeq processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Accession B15831 Members of this family are involved in a similarly diverse range of pathologies WO0042071 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The P13236/CCL4_HUMAN chemokines exert their effects by acting on a family of seven transmembrane G- C-C motif chemokine 4 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 358) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Autoimmune disorders, Immune, Vascular and Inflammatory disorders A human C-C chemokine Chemokines are a family of related small, secreted proteins involved in biological designated exodus processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies B07939 U.S. Pat. No. including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The 6,096,300 chemokines exert their effects by acting on a family of seven transmembrane G- P78556/CCL20_HUMAN protein coupled receptors. Over 40 human chemokines have been described, which C-C motif chemokine 20 bind to ~17 receptors thus far identified. Chemokine activities can be determined (isoform 1) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: (SEQ ID NO: 248) Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Cancer Human chemokine Chemokines are a family of related small, secreted proteins involved in biological L105_7 GeneSeq processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Accession Y96922 U.S. Members of this family are involved in a similarly diverse range of pathologies Pat. No. 6,084,071 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The SEQ ID NO: 4 of chemokines exert their effects by acting on a family of seven transmembrane G- WO9856818 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 437) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Chemotaxis, Gene Therapy, Wound healing Human chemokine Chemokines are a family of related small, secreted proteins involved in biological L105_3 GeneSeq processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Accession Y96923 U.S. Members of this family are involved in a similarly diverse range of pathologies Pat. No. 6,084,071 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The SEQ ID NO: 2 of chemokines exert their effects by acting on a family of seven transmembrane G- WO9856818 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 436) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Chemotaxis, Gene Therapy, Wound healing Human secondary Chemokines are a family of related small, secreted proteins involved in biological lymphoid chemokine processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. (SLC) GeneSeq Members of this family are involved in a similarly diverse range of pathologies Accession B01434 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The WO0038706 chemokines exert their effects by acting on a family of seven transmembrane G- O00585/CCL21_HUMAN protein coupled receptors. Over 40 human chemokines have been described, which C-C motif chemokine 21 bind to ~17 receptors thus far identified. Chemokine activities can be determined (SEQ ID NO: 346) using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Cancer, Vascular and Immune disorders Human non- ELR CXC Chemokines are a family of related small, secreted proteins involved in biological chemokine H174 processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies Y96310 WO0029439 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The O14625/CXL11_HUMAN chemokines exert their effects by acting on a family of seven transmembrane G- C-X-C motif chemokine 11 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 363) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune and Inflammatory disorders, Cancer, Haemostatic and thrombolytic activity Human non-ELR CXC Chemokines are a family of related small, secreted proteins involved in biological chemokine IP10 GeneSeq processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Accession Y96311 Members of this family are involved in a similarly diverse range of pathologies WO0029439 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The P02778/CXL10_HUMAN chemokines exert their effects by acting on a family of seven transmembrane G- C-X-C motif chemokine 10 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 242) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune and Inflammatory disorders, Cancer, haemostatic and thrombolytic activity Human non-ELR CXC Chemokines are a family of related small, secreted proteins involved in biological chemokine Mig GeneSeq processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Accession Y96313 Members of this family are involved in a similarly diverse range of pathologies WO0029439 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The Q07325/CXCL9_HUMAN chemokines exert their effects by acting on a family of seven transmembrane G- C-X-C motif chemokine 9 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 351) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Immune and Inflammatory disorders, Cancer, haemostatic and thrombolytic activity Human chemokine Chemokines are a family of related small, secreted proteins involved in biological Ckbeta-7 GeneSeq processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Accession Y96280 Members of this family are involved in a similarly diverse range of pathologies WO0028035 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The FIG. 1 of WO0028035 chemokines exert their effects by acting on a family of seven transmembrane G- (SEQ ID NO: 439) protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Cancer, wound healing, inflammatory and immunoregulatory disorders Human chemokine MIP- Chemokines are a family of related small, secreted proteins involved in biological 1alpha GeneSeq processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. Accession Y96281 Members of this family are involved in a similarly diverse range of pathologies WO0028035 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The P10147/CCL3_HUMAN chemokines exert their effects by acting on a family of seven transmembrane G- C-C motif chemokine 3 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 364) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Cancer, wound healing, inflammatory and immunoregulatory disorders Human mature chemokine Chemokines are a family of related small, secreted proteins involved in biological Ckbeta-7 (optionally processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. truncated) GenSeq Members of this family are involved in a similarly diverse range of pathologies Accession Y96282 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The WO0028035 chemokines exert their effects by acting on a family of seven transmembrane G- FIG. 1 of WO0028035 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 440) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Cancer, wound healing, inflammatory and immunoregulatory disorders Human chemokine Chemokines are a family of related small, secreted proteins involved in biological receptor CXCR3 GeneSeq processes ranging from Chemokine activities can be determined using assays known Accession Y79372 in the art: Methods in Molecular Biology, 2000, vol. 138: Soluble CXCR3 polypeptides WO0018431 may be useful for inhibiting P49682|CXCR3_HUMAN C-X-C chemokine receptor type 3 (isoform 1) (SEQ ID NO: 240) Human neurotactin hematopoiesis, angiogenesis, and leukocyte trafficking. Members of this family are chemokine like domain involved in a similarly diverse range of pathologies including inflammation, allergy, GeneSeq Accession tissue rejection, viral infection, and tumor biology. The chemokines exert their effects Y53259 U.S. Pat. No. by acting on a family of seven transmembrane G-protein coupled receptors. Over 40 6,043,086 human chemokines have been described, which bind to ~17 receptors thus far P78423/X3CL1_HUMAN identified. Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Fractalkine Humana Press Inc., Totowa, NJ. chemokine activities and viral infection. (SEQ ID NO: 244) Human CC type Chemokines are a family of related small, secreted proteins involved in biological chemokine interleukin C processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. GeneSeq Accession Members of this family are involved in a similarly diverse range of pathologies Y57771 JP11302298 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The chemokines exert their effects by acting on a family of seven transmembrane G- protein coupled receptors. Over 40 human chemokines have been described, which bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Cancer and infectious diseases Human CKbeta-9 Chemokines are a family of related small, secreted proteins involved in biological GeneSeq Accession processes ranging from hematopoiesis, angiogenesis, and leukocyte trafficking. B50860 U.S. Pat. No. Members of this family are involved in a similarly diverse range of pathologies 6,153,441 including inflammation, allergy, tissue rejection, viral infection, and tumor biology. The O00585/CCL21_HUMAN chemokines exert their effects by acting on a family of seven transmembrane G- C-C motif chemokine 21 protein coupled receptors. Over 40 human chemokines have been described, which (SEQ ID NO: 346) bind to ~17 receptors thus far identified. Chemokine activities can be determined using assays known in the art: Methods in Molecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E. I. Proudfoot, T. N. C. Wells, and C. A. Power. Humana Press Inc., Totowa, NJ. Cancer, Auto- immune and inflammatory disorders, Cardiovascular disorders Preproapolipo- protein Apoa-1 participates in the reverse transport of cholesterol from tissues to the liver for “paris” variant GeneSeq excretion by promoting cholesterol efflux from tissues and by acting as a cofactor for Accession W08602 the lecithin cholesterol acyltransferase (Icat). Lipid binding activity can be determined WO9637608 using assays known in the art, such as, for example, the Cholesterol Efflux Assays of (SEQ ID NO: 466) Takahaski et al., P.N.A.S., Vol. 96, Issue 20, 11358-11363, Sep. 28, 1999. Useful for cardio- vascular disorders, cholesterol disorders, and Hyperlipidaemia Preproapolipo- protein Apoa-1 participates in the reverse transport of cholesterol from tissues to the liver for “milano” variant 5,721,114 excretion by promoting cholesterol efflux from tissues and by acting as a cofactor for SEQ ID NO: 6 of U.S. the lecithin cholesterol acyltransferase (Icat). Lipid binding activity can be determined Pat. No. 5,721,114 using assays known in the art, such as, for example, the Cholesterol Efflux Assays of (SEQ ID NO: 441) Takahaski et al., P.N.A.S., Vol. 96, Issue 20, 11358-11363, Sep. 28, 1999. Useful for cardio- vascular disorders, cholesterol disorders, and Hyperlipidaemia Glycodelin-A; Naturally produced female contraceptive that is removed rapidly from the body Progesterone- associated following 2-3 days production. Uses include contraception Glycodelin-A activity can endometrial protein be determined using the hemizona assay as described in Oehninger, S., Coddington, C. C., GeneSeq Accession Hodgen, G. D., and Seppala, M (1995) Fertil. Steril. 63, 377-383. Naturally W00289 WO9628169 derived contraceptive useful for the prevention of pregnancy. P09466/PAEP_HUMAN Glycodelin (SEQ ID NO: 365) NOGO-A Genbank NOGO polypeptides are potent inhibitors of neurite growth. Inhibition of Neurite Accession CAB99248 outgrowth. Antagonists to NOGO polypeptides may promote the outgrowth of (SEQ ID NO: 366) neurites, thus inducing regeneration of neurons. NOGO-A polypep- tide antagonists are useful for the pro- motion of neural growth, which could be useful in the treatment of neural disorders and dys- function due to de- generative diseases or trauma; useful in the treatment of neo- plastic diseases of the CNS; induce regeneration of neurons or to promote the structural plasticity of the CNS. NOGO-B Genbank NOGO polypeptides are potent inhibitors of neurite growth. Inhibition of Neurite Accession CAB99249 outgrowth. Antagonists to NOGO polypeptides may promote the outgrowth of (SEQ ID NO: 367) neurites, thus inducing regeneration of neurons. NOGO-B polypep- tide antagonists are useful for the pro- motion of neural growth, which could be useful in the treatment of neural disorders and dys- function due to de- generative diseases or trauma; useful in the treatment of neo- plastic diseases of the CNS; induce regeneration of neurons or to promote the structural plasticity of the CNS. NOGO-C Genbank NOGO polypeptides are potent inhibitors of neurite growth. Inhibition of Neurite Accession CAB99250 outgrowth. Antagonists to NOGO polypeptides may promote the outgrowth of (SEQ ID NO: 368) neurites, thus inducing regeneration of neurons. NOGO-C polypep- tide antagonists are useful for the pro- motion of neural growth, which could be useful in the treatment of neural disorders and dys- function due to de- generative diseases or trauma; useful in the treatment of neo- plastic diseases of the CNS; induce regeneration of neurons or to promote the structural plasticity of the CNS. NOGO-66 Receptor NOGO polypeptides are potent inhibitors of neurite growth, and are thought to Genbank Accession mediate their effects through the NOGO-66 Receptor. Inhibition of Neurite outgrowth AAG53612 by mediating the biological effects of NOGO polypeptides. Soluble NOGO- 66 (SEQ ID NO: 369) receptor polypeptides may promote the outgrowth of neurites, thus inducing regeneration of neurons. NOGO-66 receptor polypeptides are useful for the promotion of neural growth, which could be useful in the treatment of neural disorders and dys- function due to de- generative diseases or trauma; useful in the treatment of neo- plastic diseases of the CNS; induce regeneration of neurons or to promote the structural plasticity of the CNS. Antibodies specific for These antibodies are useful for the promotion of neurite outgrowth Collapsin activity, collapsin U.S. Pat. No. which is thought to inhibit the outgrowth of neurites, can be assayed in the presence 5,416,197 of antibodies specific for collapsing using assays known in the art, such as, for Wildtype collapsin has the example, the collapse assay disclosed by Luo et al., Cell 1993 Oct. 22; 75(2): 217-27 sequence: Useful for the pro- motion of neural growth, which could be useful in the treatment of SEQ ID NO: 2 of 5,416,197 neural disorders and dys- function due to de- generative diseases or trauma. (SEQ ID NO: 464) Humanized Anti-VEGF These agents have anti-inflammatory and anti-cancer applications VEGF activity can Antibodies, and fragments be determined using assays known in the art, such as those disclosed in International thereof WO9845331 Publication No. WO0045835, for example. Promotion of growth and proliferation of cells, such as vascular endothelial cells. Antagonists may be useful as anti- angiogenic agents, and may be applicable for cancer Humanized Anti-VEGF These agents have anti-inflammatory and anti-cancer applications VEGF activity can Antibodies, and fragments be determined using assays known in the art, such as those disclosed in International thereof WO0029584 Publication No. WO0045835, for example. Promotion of growth and proliferation of cells, such as vascular endothelial cells. Hematopoietic and immune dis- orders. Antagonists may be useful as anti-angiogenic agents, and may be applicable for cancer Membrane bound proteins Cancer, Immune Disorders These proteins can be used for linking bioactive GeneSeq. Accession molecules to cells and for modulating biological activities of cells, using the Y66631-Y66765 polypeptides for specific targeting. The polypeptide targeting can be used to kill the WO9963088 target cells, e.g. for the treatment of cancers. These proteins are useful for the treatment of immune system disorders. Activities can be determined using assay known in the art, such as, for example, the assays disclosed in International Publication No. WO0121658. Secreted and Cancer, Immune Disorders These proteins can be used for linking bioactive Transmembrane molecules to cells and for modulating biological activities of cells, using the polypeptides GeneSeq polypeptides for specific targeting. The polypeptide targeting can be used to kill the Accession B44241-B44334 target cells, e.g. for the treatment of cancers. These proteins are useful for the WO0053756 treatment of immune system disorders. Activities can be determined using assay known in the art, such as, for example, the assays disclosed in International Publication No. WO0121658. Secreted and Cancer, Immune Disorders These proteins can be used for linking bioactive Transmembrane molecules to cells and for modulating biological activities of cells, using the polypeptides GeneSeq polypeptides for specific targeting. The polypeptide targeting can be used to kill the Accession Y41685-Y41774 target cells, e.g. for the treatment of cancers. These proteins are useful for the WO9946281 treatment of immune system disorders. Activities can be determined using assay known in the art, such as, for example, the assays disclosed in International Publication No. WO0121658. Interleukin 2 (IL-2) Metabolic Disease, Type 1 diabetes, Graft-versus-host disease. SEQ ID NO: 548 Interleukin 15_vA Obesity, Metabolic Disease, Diabetes. (IL-15_vA) SEQ ID NO: 549 Interleukin 15_vB Obesity, Metabolic Disease, Diabetes. (IL-15_vB) SEQ ID NO: 550 Interleukin 15_vC Obesity, Metabolic Disease, Diabetes. (IL-15_vC) SEQ ID NO: 551 Interleukin 15_vD Obesity, Metabolic Disease, Diabetes. (IL15_vD) SEQ ID NO: 552 Interleukin 15_vE Obesity, Metabolic Disease, Diabetes. (IL15_vE) SEQ ID NO: 553 Interleukin 15_vF Obesity, Metabolic Disease, Diabetes. (IL15_vF) SEQ ID NO: 565 Interleukin 22 Metabolic Disease, Diabetic Ulcers, Inflamatory Bowel Diseases. (IL22) SEQ ID NO: 554 Fibroblast Growth Factor 1 Diabetes, Metabolic Disease, Obesity. (FGF1) SEQ ID NO: 555 Fibroblast Growth Factor Diabetes, Metabolic Disease, Obesity. 1_vA See Nature 513, 436-439 (18 Sep. 2014) doi: 10.1038/nature13540. (FGF1_vA) SEQ ID NO: 556 Fibroblast Growth Factor Diabetes, Metabolic Disease, Obesity. 1_vB See Proc Natl Acad Sci USA. 1991 Apr. 1; 88(7): 2893-2897. (FGF1_vB) SEQ ID NO: 557 Fibroblast Growth Factor Diabetes, Metabolic Disease, Obesity. 1_vC (FGF1_vC) SEQ ID NO: 566 Fibroblast Growth Factor Chronic liver disease, primary biliary cirrhosis, bile acid-induced liver damage. 19_vA See Cancer Res. 2014 Jun. 15; 74(12): 3306-16. doi: 10.1158/0008-5472.CAN-14- (FGF19_vA) 0208. Epub 2014 Apr. 11. Regulates bile acid metabolism without tumorigenicity. SEQ ID NO: 558 Fibroblast Growth Factor Metabolic Disease, Fibrotic Diseases. 21 (FGF21) SEQ ID NO: 559 Fibroblast Growth Factor Hyperphosphatemic familial tumoral calcinosis. 23 (FGF23) SEQ ID NO: 560 Brain-Derived Neurological diseases (including Alzheimer's Disease, Autism, Huntington's Disease, Neurotrophic Factor Parkinson's Disease, and Depression), obesity, metabolic disease. (BDNF) SEQ ID NO: 561 Serpin Family A Member 1 alpha-1-antitrypsin deficiency. (SERPINA1) SEQ ID NO: 584 SEQ ID NO: 585 Serpin Peptidase Inhibitor, Diabetes. Clade B (Ovalbumin), Member 1 (SERPINB1) SEQ ID NO: 562 CASPASE1 Diabetes. SEQ ID NO: 563 Leukemia Inhibitory Factor Muscular dystrophy, atherosclerosis, kidney disease (LIF) SEQ ID NO: 564 Proprotein Convertase Cardiovascular disease, hypercholesterolemia, heterozygous familial Subtilisin/Kexin Type 1 hypercholesterolemia (HeFH), atherosclerotic cardiovascular disease such as heart (PCSK1) attacks or strokes, increasing the amount of a functional protein, polypeptide or SEQ ID NO: 567 peptide Proprotein Convertase Cardiovascular disease, hypercholesterolemia, heterozygous familial Subtilisin/Kexin Type 2 hypercholesterolemia (HeFH), atherosclerotic cardiovascular disease such as heart (PCSK2) attacks or strokes, increasing the amount of a functional protein, polypeptide or SEQ ID NO: 568 peptide Proprotein Convertase Cardiovascular disease, hypercholesterolemia, heterozygous familial Subtilisin/Kexin Type 3 hypercholesterolemia (HeFH), atherosclerotic cardiovascular disease such as heart (PCSK3) attacks or strokes, increasing the amount of a functional protein, polypeptide or SEQ ID NO: 569 peptide Proprotein Convertase Cardiovascular disease, hypercholesterolemia, heterozygous familial Subtilisin/Kexin Type 3 Sol hypercholesterolemia (HeFH), atherosclerotic cardiovascular disease such as heart (PCSK3_SOL) attacks or strokes, increasing the amount of a functional protein, polypeptide or SEQ ID NO: 570 peptide Proprotein Convertase Cardiovascular disease, hypercholesterolemia, heterozygous familial Subtilisin/Kexin Type 4 hypercholesterolemia (HeFH), atherosclerotic cardiovascular disease such as heart (PCSK4) attacks or strokes, increasing the amount of a functional protein, polypeptide or SEQ ID NO: 571 peptide Proprotein Convertase Cardiovascular disease, hypercholesterolemia, heterozygous familial Subtilisin/Kexin Type 5 hypercholesterolemia (HeFH), atherosclerotic cardiovascular disease such as heart (PCSK5) attacks or strokes, increasing the amount of a functional protein, polypeptide or SEQ ID NO: 572 peptide Proprotein Convertase Cardiovascular disease, hypercholesterolemia, heterozygous familial Subtilisin/Kexin Type 6 hypercholesterolemia (HeFH), atherosclerotic cardiovascular disease such as heart (PCSK6) attacks or strokes, increasing the amount of a functional protein, polypeptide or SEQ ID NO: 573 peptide Proprotein Convertase Cardiovascular disease, hypercholesterolemia, heterozygous familial Subtilisin/Kexin Type hypercholesterolemia (HeFH), atherosclerotic cardiovascular disease such as heart (PCSK7) attacks or strokes, increasing the amount of a functional protein, polypeptide or SEQ ID NO: 574 peptide Proprotein Convertase Cardiovascular disease, hypercholesterolemia, heterozygous familial Subtilisin/Kexin Type 8 hypercholesterolemia (HeFH), atherosclerotic cardiovascular disease such as heart (PCSK8) attacks or strokes, increasing the amount of a functional protein, polypeptide or SEQ ID NO: 575 peptide Proprotein Convertase Cardiovascular disease, hypercholesterolemia, heterozygous familial Subtilisin/Kexin Type 9 hypercholesterolemia (HeFH), atherosclerotic cardiovascular disease such as heart (PCSK9) attacks or strokes, increasing the amount of a functional protein, polypeptide or SEQ ID NO: 576 peptide Membrane-Bound IFAP syndrome, increasing the amount of a functional protein, polypeptide or peptide Transcription Factor Peptidase, Site 2 (MBTPS2) SEQ ID NO: 577 Carboxypeptidase E Endocrine disorders, such as, for example, obesity and infertility; (CPE) hyperproinsulinemia; metabolic syndrome, increasing the amount of a functional SEQ ID NO: 578 protein, polypeptide or peptide

In various embodiments, the nucleic acid drug, including synthetic RNA, is administered is a manner that it effects one or more of keratinocytes and fibroblasts (e.g. causes these cells to express one or more therapeutic proteins). For example, present methods allow for methods in which a patient's cells are used to generate a therapeutic protein and the levels of such protein are tailored by synthetic RNA dosing.

In a specific embodiment, the synthetic RNA targets a soluble protein. In some embodiments, the synthetic RNA targets a protein of one or more of the following families of proteins: transforming growth factor (TGF) beta, bone morphogenetic proteins (BMPs), Fibroblast growth factors (FGFs), vascular endothelial growth factors (VEGFs), and interleukins. The terms “family”, “superfamily”, and “subfamily” can be used interchangeably

In a specific embodiment, the synthetic RNA targets a member of the TGF beta family. TGF-β superfamily proteins comprise cytokines characterized by six-conserved cysteine residues (Lander et al., (2001) Nature, 409:860-921). The human genome contains at least about 42 open reading frames encoding TGF-β superfamily proteins. TGF-β superfamily proteins can at least be divided into the BMP subfamily and the TGF-β subfamily based on sequence similarity and the specific signaling pathways that they activate. In various embodiments, the synthetic RNA targets one or more of TGFs (e.g., TGF-β1, TGF-β2, and TGF-β3), activins (e.g., activin A) and inhibins, macrophage inhibitory cytokine-1 (MIC-1), Mullerian inhibiting substance, anti-Mullerian hormone, and glial cell line derived neurotrophic factor (GDNF).

The TGF-β superfamily comprises a subset of the cysteine knot cytokine superfamily. Additional members of the cysteine knot cytokine superfamily include, but are not limited to, platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), placenta growth factor (P1GF), Noggin, neurotrophins (BDNF, NT3, NT4, and PNGF), gonadotropin, follitropin, lutropin, interleukin-17, and coagulogen. This family of proteins is also among the targets encompassed by the present invention.

In various embodiments, the present invention relates to targeting a TGF beta family member for treatment or prevention of various immunological disorders, cancer, bronchial asthma, lung fibrosis, heart disease, diabetes, hereditary hemorrhagic telangiectasia, Marfan syndrome, Vascular Ehlers-Danlos syndrome, Loeys-Dietz syndrome, Parkinson's disease, chronic kidney disease, multiple sclerosis and AIDS.

In a specific embodiment, the synthetic RNA targets a member of the BMP family. The BMP subfamily includes, but is not limited to, BMP-2, BMP-3 (osteogenin), BMP-3b (GDF-10), BMP-4 (BMP-2b), BMP-5, BMP-6, BMP-7 (osteogenic protein-1 or OP-1), BMP-8 (OP-2), BMP-8B (OP-3), BMP-9 (GDF-2), BMP-10, BMP-11 (GDF-11), BMP-12 (GDF-7), BMP-13 (GDF-6, CDMP-2), BMP-15 (GDF-9), BMP-16, GDF-1, GDF-3, GDF-5 (CDMP-1), and GDF-8 (myostatin). In various embodiments, the synthetic RNA targets one or more of BMP-2, BMP-3 (osteogenin), BMP-3b (GDF-10), BMP-4 (BMP-2b), BMP-5, BMP-6, BMP-7 (osteogenic protein-1 or OP-1), BMP-8 (OP-2), BMP-8B (OP-3), BMP-9 (GDF-2), BMP-10, BMP-11 (GDF-11), BMP-12 (GDF-7), BMP-13 (GDF-6, CDMP-2), BMP-15 (GDF-9), BMP-16, GDF-1, GDF-3, GDF-5 (CDMP-1), and GDF-8 (myostatin). BMPs are sometimes referred to as Osteogenic Protein (OPs), Growth Differentiation Factors (GDFs), or Cartilage-Derived Morphogenetic Proteins (CDMPs). In a specific embodiment, the synthetic RNA targets one or more BMP fusions (e.g. as described in US Patent Publication No. 2009/0202638, the entire contents of which are hereby incorporated by reference) and/or one or more BMP mutants (e.g. as described in US Patent Publication No. 2011/0039773, the entire contents of which are hereby incorporated by reference).

In various embodiments, the present invention relates to targeting a BMP family member for regenerative medicine or metabolic applications, including without limitation orthopedic applications such as spinal fusions, nonunions and oral surgerie, metabolic disease, prediabetes, diabetes, thermogenesis, insulin sensitivity, insulin resistance, and adipogenesis, including brown-fat adipogenesis. Various other metabolic applications are described elsewhere herein. In various embodiments, the present invention relates to targeting a BMP family member, including BMP-7, for treatment of chronic kidney disease (CKD) and/or to reverse the loss of glomeruli due to sclerosis.

In various embodiments, the present invention relates to targeting a BMP family member to induce proliferation of bone and cartilage in a variety of locations in the body. For example, repair of joints such as knee, elbow, ankle, and finger joints are contemplated by the invention. For example, targeting a BMP family member may result in regenerating cartilage in patients suffering from arthritis or other cartilage degenerating diseases. Further, the invention pertains to treating tears in cartilage due to injury. In addition, the invention is useful for inducing bone growth in patients, for instance, by way of non-limitation, for use in treating patients suffering from bone fractures or breaks, osteoporosis, or patients in need of spinal fusion or for repair of the spine, vertebrae or the like.

In various embodiments, the present invention relates to targeting a BMP family member to induce a developmental cascade of bone morphogenesis and tissue morphogenesis for a variety of tissues in mammals different from bone or bone cartilage. This morphogenic activity includes the ability to induce proliferation and differentiation of progenitor cells, and the ability to support and maintain the differentiated phenotype through the progression of events that results in the formation of bone, cartilage, non-mineralized skeletal or connective tissues, and other adult tissues.

For example, the present invention may be used for treatment to prevent loss of and/or increase bone mass in metabolic bone diseases. General methods for treatment to prevent loss of and/or increase bone mass in metabolic bone diseases using osteogenic proteins are disclosed in U.S. Pat. No. 5,674,844, the entire contents of which are hereby incorporated by reference. Further the present compositions and methods find use in replacing or repairing bone or cartilage at injury sites such as bone breaks, bone fractures, and cartilage tears, periodontal tissue regeneration (e.g. general methods for periodontal tissue regeneration using osteogenic proteins are disclosed in U.S. Pat. No. 5,733,878, the entire contents of which are hereby incorporated by reference), liver regeneration, including following a partial hepatectomy (see, e.g., U.S. Pat. No. 5,849,686, the entire contents of which are hereby incorporated by reference), treatment of chronic renal failure (see, e.g., U.S. Pat. No. 6,861,404, the entire contents of which are hereby incorporated by reference), enhancing functional recovery following central nervous system ischemia or trauma (see, e.g. U.S. Pat. No. 6,407,060, the entire contents of which are hereby incorporated by reference), inducing dendritic growth (see, e.g., U.S. Pat. No. 6,949,505, the entire contents of which are hereby incorporated by reference), inducing neural cell adhesion (see, e.g., U.S. Pat. No. 6,800,603, the entire contents of which are hereby incorporated by reference), and treatment and prevention of Parkinson's disease (see, e.g., U.S. Pat. No. 6,506,729, the entire contents of which are hereby incorporated by reference). As another example, the present compositions and methods, including when targeting one or more BMPs, can be used to induce dentinogenesis. To date, the unpredictable response of dental pulp tissue to injury is a basic clinical problem in dentistry. Using standard dental surgical procedures, small areas (e.g., 2 mm) of dental pulps can be surgically exposed by removing the enamel and dentin immediately above the pulp (by drilling) of sample teeth, performing a partial amputation of the coronal pulp tissue, inducing hemostasis, application of the pulp treatment, and sealing and filling the cavity by standard procedures.

In various embodiments, the present invention relates to targeting a BMP family member for the treatment of one or more metabolic-related disorders as described herein.

In a specific embodiment, the synthetic RNA targets a member of the FGF family. FGFs are a family of growth factors, with members involved in angiogenesis, wound healing, embryonic development and various endocrine signaling pathways. In various embodiments, any one of the following FGFs are targets of the invention: FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14 (FGF11, FGF12, FGF13, and FGF14 being FGF homologous factors 1-4 (FHF1-FHF4)), FGF16, FGF17, FGF18, FGF19 (aka FHF15/19), FGF20, FGF21, FGF22 and FGF22. In some embodiments, the synthetic RNA targets a cognate receptor of any of the above FGFs. In various embodiments, the present invention relates to targeting a FGF family member to a treat or prevent disease or disorder associated with abnormal function and/or expression of an FGF, a metabolic disease or disorder (e.g., diabetes, obesity, dyslipidemia, hyperglycemia, hyperinsulinemia, hypertension, hepatosteaotosis such as non-alcoholic steatohepatitis (NASH) etc.), cancer, a disease or disorder associated with impaired lipid metabolism a disease or disorder associated with impaired renal function, a disease or disorder associated with impaired hepatic function, abnormal cell proliferation, a vascular disease or disorder (e.g., coronary artery disease, peripheral artery disease, atherosclerosis, abdominal aortic aneurysm, a blood clot, deep vein thrombosis, venous stasis disease, phlebitis, varicose veins etc.), angiogenesis, atherosclerosis, a cardiovascular disease or disorder, a disease or disorder associated with impaired blood vessel formation, a disease or disorder associated with impaired cell signaling, a disease or disorder associated with impaired kinase activity, and a disease or disorder associated with impaired uptake of glucose into adipocytes.

In a specific embodiment, the synthetic RNA targets a member of the VEGF family. In some embodiments, the target is one or more of VEGF-A (including all isoforms, e.g. VEGF121, VEGF165 and VEGF189), placenta growth factor (PGF, including all isoforms, e.g. PGF-1, PGF-2, and PGF-3), VEGF-B, VEGF-C and VEGF-D, and any variants thereof (see, e.g. U.S. Pat. No. 9,078,860, the entire contents of which are hereby incorporated by reference). In some embodiments, the synthetic RNA targets a cognate receptor of any of the above VEGFs. The present invention also encompasses as targets VEGF-related proteins including orf virus-encoded VEGF-like proteins referred to as VEGF-E and a series of snake venoms referred to as VEGF-F. VEGFs and VEGF-related proteins are members of the Platelet Derived Growth Factor (PDGF) supergene family of cystine knot growth factors. All members of the PDGF supergene family share a high degree of structural homology with PDGF.

In various embodiments, the present invention relates to targeting a VEGF family member to treat diseases and conditions associated with angiogenesis, including but not limited to, solid tumor cancers, hemangiomas, rheumatoid arthritis, osteoarthritis, septic arthritis, asthma, atherosclerosis, idiopathic pulmonary fibrosis, vascular restenosis, arteriovenous malformations, meningiomas, neovascular glaucoma, psoriasis, Kaposi's Syndrome, angiofibroma, hemophilic joints, hypertrophic scars, Osler-Weber syndrome, pyogenic granuloma, retrolental fibroplasias, scleroderma, trachoma, von Hippel-Lindau disease, vascular adhesion pathologies, synovitis, dermatitis, neurological degenerative diseases, preeclampsia, unexplained female infertility, endometriosis, unexplained male infertility, pterygium, wounds, sores, skin ulcers, gastric ulcers, and duodenal ulcers. In various embodiments, the present invention relates to targeting a VEGF family member to treat angiogenesis-associated eye diseases, including without limitation any eye disease associated with abnormal intraocular neovascularization, including but not limited to retinopathy of prematurity, diabetic retinopathy, retinal vein occlusion, and age-related macular degeneration, as well diabetic macular edema and retinal vein occlusion. In an embodiment, the present compositions and methods relate to the treatment of wet age-related macular degeneration.

In a specific embodiment, the synthetic RNA targets a member of the interleukin family. The interleukins represent a large group of cytokines with diverse functions and were first characterized by expression in leukocytes and have since been shown to be expressed in a wide variety of cells, for example macrophages, TH-1 and TH-2 cells, T-lymphocytes, monocytes and bone marrow stroma. Broadly, the function of the immune system depends in a large part on the expression and function of the interleukins. In some embodiments, the target is one or more of interleukins 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35 and 36, and, within each species of interleukin, various isotypes and/or interleukin receptors (e.g., IL-1R, IL-2R, IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-8R, IL-9R, IL-10R, IL-11R, IL-12R, IL-13R, IL-14R, IL-15R, IL-16R, IL-17R, IL-18R, IL-19R, IL-20R, IL-21R, IL-22R, IL-23R, IL-24R, IL-25R, IL-26, IL-27R, IL-28R, IL-29R, IL-30R, IL-31R, IL-32R, IL-33R, IL-34R, IL-35R, and IL-36R). In a specific embodiment, both IL-15 and IL-15R (e.g., IL-15RA) are targeted. Without wishing to be bound by theory, it is believed that the targeting of both interleukin (e.g., IL-15) and its cognitive interleukin receptor (e.g., IL-15RA) results in synergistic beneficial effects. In various embodiments, the present invention relates to targeting a member of the interleukin family to treat cancer, inflammatory, respiratory, autoimmune, cardiovascular, neurological, metabolic, and/or proliferative diseases, disorders, and/or conditions in a subject or organism, as described herein. In a specific embodiment, the present invention relates to targeting a member of the interleukin family to treat cancer. In a specific embodiment, the present invention relates to targeting a member of the interleukin family to treat rheumatoid arthritis.

In a specific embodiment, the synthetic RNA targets an EPO gene or a derivative thereof (e.g. SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, and SEQ ID NO: 167). Some embodiments are related to NOVEPOETIN protein (SEQ ID NO: 167). Other embodiments are related to NOVECRIT (SEQ ID NO: 168).

Erythropoietin can stimulate erythropoiesis in anemic patients with chronic renal failure in whom the endogenous production of erythropoietin is impaired. Without being bound by theory, because of the length of time often required for erythropoiesis (several days for erythroid progenitors to mature and be released into the circulation), a clinically significant increase in hemoglobin is usually not observed in less than two weeks and may require up to ten weeks in some patients. The present methods and compositions provide, in some embodiments, more rapid therapeutic effect. The present methods and compositions provide, in some embodiments, more rapid therapeutic effect, for instance, when compared to wild type EPO and/or EPO without non-canonical nucleotides and/or EPO delivered as a protein biologic. The present methods and compositions provide, in some embodiments, a sustained therapeutic effect. The present methods and compositions provide, in some embodiments, a sustained therapeutic effect, for instance, when compared to wild type EPO and/or EPO without non-canonical nucleotides and/or EPO delivered as a protein biologic.

For instance, for EPO, the present methods and compositions provide a clinically significant increase in hematocrit in less than about 6 weeks, or less than about 5 weeks, or less than about 4 weeks, or less than about 3 weeks, or less than about 2 weeks, or less than about 1 week. In some embodiments, the present methods and compositions provide a clinically significant increase in hematocrit in about 2 weeks, or about 10 days, or about 1 week, or about 3 days, or about 1 day. In various embodiments, the present methods and compositions accelerate the process by which erythroid progenitors mature and are released into the circulation.

In some embodiments, the present EPO-related compositions find use in decreasing the dose and/or frequency of administration when compared to wild type EPO and/or EPO without non-canonical nucleotides and/or EPO delivered as a protein biologic. For instance, the present EPO-related compositions may find use in treatment regimens for the diseases disclosed herein (including, without limitation, one or more anemias) that involve administration on a monthly, or biweekly, or weekly basis. In some embodiments, therefore, the present EPO-related compositions reduce the need for daily, or, in some embodiments, weekly, administration. In some embodiments, the present EPO-related compositions require lower maintenance doses as compared to wild type EPO and/or EPO without non-canonical nucleotides and/or EPO delivered as a protein biologic. Certain embodiments are particularly useful for treating chemotherapy-induced anemia. Other embodiments are particularly useful for treating anemia associated with inflammation, including, but not limited to, rheumatoid arthritis.

In some embodiments, the present the present methods and compositions provide a fast and robust response that obviates the need for RBC transfusion. For instance, in some embodiments, the present methods and compositions allow for treatment of patients do not consent to transfusions.

In some embodiments, the present methods and compositions increase the rate of increase in hematocrit. In some embodiments, the present methods and compositions maintain elevated hematocrits (e.g. of 25%, or 30%, or 35%, or 40% or more) for a sustained period (e.g. about 1 month, or about 2 months, or about 3 months, or about 4 months, or about 5 months, or about 6 months, or about 9 months).

In some embodiments, the present methods and compositions stimulate red blood cell production. In some embodiments, the present methods and compositions stimulate division and differentiation of committed erythroid progenitors in the bone marrow.

In some embodiments, including, without limitation, when targeting EPO, the present invention relates to the treatment of one or more of anemia, including anemia resulting from resulting from chronic kidney disease (e.g. from dialysis) and/or chemotherapy and/or HIV treatment (e.g. Zidovudine (INN) or azidothymidine (AZT)), inflammatory bowel disease (e.g. Crohn's disease and ulcer colitis), anemia linked to inflammatory conditions (e.g. arthritis, lupus, IBD), anemia linked to diabetes, schizophrenia, cerebral malaria, as aplastic anemia, and myelodysplasia from the treatment of cancer (e.g. chemotherapy and/or radiation), and various myelodysplastic syndrome diseases (e.g. sickle cell anemia, hemoglobin SC disease, hemoglobin C disease, alpha- and beta-thalassemias, neonatal anemia after premature birth, and comparable conditions).

In some embodiments, including, without limitation, when targeting EPO, the present invention relates to the treatment of, or a patient having one or more of cancer, heart failure, autoimmune disease, sickle cell disease, thalassemia, blood loss, transfusion reaction, diabetes, vitamin B12 deficiency, collagen vascular disease, Shwachman syndrome, thrombocytopenic purpura, Celiac disease, endocrine deficiency state such as hypothyroidism or Addison's disease, autoimmune disease such as Crohn's Disease, systemic lupus erythematosis, rheumatoid arthritis or juvenile rheumatoid arthritis, ulcerative colitis immune disorders such as eosinophilic fasciitis, hypoimmunoglobulinemia, or thymoma/thymic carcinoma, graft vs. host disease, preleukemia, Nonhematologic syndrome (e.g. Down's, Dubowwitz, Seckel), Felty syndrome, hemolytic uremic syndrome, myelodysplasic syndrome, nocturnal paroxysmal hemoglobinuria, osteomyelofibrosis, pancytopenia, pure red-cell aplasia, Schoenlein-Henoch purpura, malaria, protein starvation, menorrhagia, systemic sclerosis, liver cirrhosis, hypometabolic states, congestive heart failure, chronic infections such as HIV/AIDS, tuberculosis, oseomyelitis, hepatitis B, hepatitis C, Epstein-barr virus or parvovirus, T cell leukemia virus, bacterial overgrowth syndrome, fungal or parasitic infections, and/or red cell membrane disorders such as hereditary spherocytosis, hereditary elliptocytosis, heriditray pyrpoikilocytosis, hereditary stomatocytosis, red cell enzyme defects, hypersplenism, immune hemolysis or paroxysmal nocturnal hemoglobinuria.

In some embodiments, including, without limitation, when targeting EPO, the present invention relates to the treatment of, or a patient having anemia, i.e. a condition in which the number of red blood cells and/or the amount of hemoglobin found in the red blood cells is below normal. In various embodiments, the anemia may be acute or chronic. For example, the present anemias include but are not limited to iron deficiency anemia, renal anemia, anemia of chronic diseases/inflammation, pernicious anemia such as macrocytic achylic anemia, juvenile pernicious anemia and congenital pernicious anemia, cancer-related anemia, chemotherapy-related anemia, radiotherapy-related anemia, pure red cell aplasia, refractory anemia with excess of blasts, aplastic anemia, X-lined siderobalstic anemia, hemolytic anemia, sickle cell anemia, anemia caused by impaired production of ESA, myelodysplasia syndromes, hypochromic anemia, microcytic anemia, sideroblastic anemia, autoimmune hemolytic anemia, Cooley's anemia, Mediterranean anemia, Diamond Blackfan anemia, Fanconi's anemia and drug-induced immune hemolytic anemia. Anemia may cause serious symptoms, including hypoxia, chronic fatigue, lack of concentration, pale skin, low blood pressure, dizziness and heart failure.

In some embodiments, the anemia is induced by chemotherapy is one cause of anemia. For instance, the chemotherapy may be any myelosuppressive chemotherapy. In some embodiments, the chemotherapy is one or more platinum-based drugs including cisplatin (e.g. PLATINOL) and carboplatin (e.g. PARAPLATIN). In some embodiments, the chemotherapy is any one of the agents described herein. In some embodiments, the chemotherapy is any agent described in Groopman et al. J Natl Cancer Inst (1999) 91 (19): 1616-1634, the contents of which are hereby incorporated by reference in their entireties. In some embodiments, the present compositions and methods are used in the treatment of chemotherapy-related anemia in later stage cancer patients (e.g. a stage IV, or stage Ill, or stage II cancer). In some embodiments, the present compositions and methods are used in the treatment of chemotherapy-related anemia in cancer patients receiving dose-dense chemotherapy or other aggressive chemotherapy regimens.

In some embodiments, the present invention relates to the treatment of anemia in a patient having one or more blood-based cancers, such as leukemia, lymphoma, and multiple myeloma. Such cancers may affect the bone marrow directly. Further, the present invention relates to metastatic cancer that has spread to the bone or bone marrow. In some embodiments, the present invention relates to the treatment of anemia in a patient undergoing radiation therapy. Such radiation therapy may damage the bone marrow, lowering its ability to make red blood cells. In further embodiments, the present invention relates to the treatment of anemia in a patient having a reduction or deficiency of one or more of iron, vitamin B12, and folic acid. In further embodiments, the present invention relates to the treatment of anemia in a patient having excessive bleeding including without limitation, after surgery or from a tumor that is causing internal bleeding. In further embodiments, the present invention relates to the treatment of anemia in a patient having anemia of chronic disease.

In some embodiments, the present invention relates to the treatment of anemia resulting from chronic renal failure. In some embodiments, the present invention relates to the treatment of anemia resulting from the use of one or more renal replacement therapies, inclusive of dialysis, hemodialysis, peritoneal dialysis, hemofiltration, hemodiafiltration, and renal transplantation.

In some embodiments, the present invention relates to the treatment of anemia in patients with chronic kidney disease who are not on dialysis. For instance, the present invention relates to patients in stage 1 CKD, or stage 2 CKD, or stage 3 CKD, or stage 4 CKD, or stage 5 CKD. In some embodiments, the present patient is stage 4 CKD or stage 5 CKD. In some embodiments, the present patient has undergone a kidney transplant. In some embodiments, the present invention relates to the treatment of anemia is a patient having an acute kidney injury (AKI).

In various embodiments, the present compositions and methods are used to reduce or eliminate fatigue, dizziness, and shortness of breath in a patient.

In various embodiments, the present compositions and methods are used to treat a patient presenting with hyporesponse or resistance to erythropoiesis stimulating agent therapy. In some embodiments, hyporesponsiveness to erythropoietin or ESA-resistant anemia refers to the presence of at least one of the following conditions: i) a significant decrease in hemoglobin levels at a constant dose of ESA treatment, ii) a significant increase in the ESA dose requirement to achieve or maintain a certain hemoglobin level, iii) a failure to raise the hemoglobin level to the target range despite the ESA dose equivalent to erythropoietin greater than 150 IlU/kg/week or 0.75 mg/kg/week of darbepoeitn-alpha or continued need for such high dose of ESA to maintain the target hemoglobin level. For example, approximately 5-10% of patients with CDK demonstrate hyporesponsiveness to ESA, defined as a continued need for greater than 300 IU/kg per week erythropoietin or 1.5 ng/kg per week darbepoetin administered by the subcutaneous route.

In various embodiments, the present compositions and methods mitigate the need for dose-escalation of the erythropoiesis stimulating agent therapy and therefore, optionally, avoid side effects (e.g. flu-like symptoms such as joint pains, weakness, dizziness and tiredness, skin irritation, increased risk of adverse cardiovascular complications).

In various embodiments, the present compositions and methods are used to maintain a hemoglobin level of about 12.5 to 13 g/dL. In various embodiments, the present compositions and methods are used in patients having hemoglobin levels of below about 12 g/dL, or about 11 g/dL, or about 10 g/dL, or about 9 g/dL, or about 8 g/dL, or about 7 g/dL, or about 6 g/dL, or about 5 g/dL. In various embodiments, the present compositions and methods are used in patients having iron blood test scores that indicate blood pathology, e.g. a ferritin score of below about 200 ng/L and/or a transferrin saturation score below about 30%.

In various embodiments, the present compositions and methods are used to increase or maintain hemoglobin levels at a target level ranging from 9 to 10 g/dL, at a target level ranging from 9 g/dL to 11 g/dL, at a target level ranging from 9 g/dL to 12 g/dL, at a target level ranging from 9 g/dL to 14 g/dL, at a target level ranging from 10 g/dL to 14 g/dL, or at a target level ranging from 12 g/dL to 14 g/dL.

In various embodiments, the present compositions and methods are used to bring a patient's hemoglobin levels to normal. In various embodiments, normal hemoglobin ranges for humans are about 14-18 g/dl for men and 12-16 for women g/dl with the average hemoglobin value for men at about 16 g/dL and for women at about 14 g/dL.

In some embodiments, for instance when targeting EPO, the present invention relates to the treatment of anemia of one or more of the following toxicity grading criteria (e.g. NCI Common Toxicity Criteria): grade 1 (mild), 10.0 g hemoglobin/dL to within normal limits; grade 2 (moderate), 8.0-10.0 g of hemoglobin/dL; grade 3 (serious or severe), 6.5-7.9 g of hemoglobin/dL; and grade 4 (life threatening), less than 6.5 g of hemoglobin/dL. In various embodiments, the present invention brings an increase in toxicity grading criteria by about 1 point, or about 2 points, or about 3 points, or about 4 points. In various embodiments, the present invention results in a patient having a level of 0 or 1. In various embodiments, the present compositions and methods improve anemia as assessed by one or more scales described in Groopman et al. J Natl Cancer Inst (1999) 91 (19): 1616-1634, the entire contents of which are hereby incorporated by reference in their entireties.

In some embodiments, when targeting EPO, the present invention relates to combination therapy with one or more EPOs. For example, the present compositions may provide a sustained effect that can supplement a fast action of another EPO. In some embodiments, the present compositions are used as an adjuvant to other EPOs. In some embodiments, the present compositions are used as a maintenance therapy to other EPOs Other EPOs include the following: epoetin alfa, including without limitation, DARBEPOETIN (ARANESP)_EPOCEPT (LUPIN PHARMA)_NANOKINE (NANOGEN PHARMACEUTICAL), EPOFIT (INTAS PHARMA), EPOGEN (AMGEN), EPOGIN, EPREX, (JANSSEN-CILAG), BINOCRIT (SANDOZ), PROCRIT; epoetin beta, including without limitation, NEORECORMON (HOFFMANN-LA ROCHE), RECORMON, Methoxy polyethylene glycol-epoetin beta (MIRCERA, ROCHE); epoetin delta, including without limitation, DYNEPO (erythropoiesis stimulating protein, SHIRE PLC); epoetin omega, including without limitation, EPOMAX; epoetin zeta, including without limitation, SILAPO (STADA) and RETACRIT (HOSPIRA) and other EPOs, including without limitation, EPOCEPT (LUPIN PHARMACEUTICALS), EPOTRUST (PANACEA BIOTEC LTD), ERYPRO SAFE (BIOCON LTD.), REPOITIN (SERUM INSTITUTE OF INDIA LIMITED), VINTOR (EMCURE PHARMACEUTICALS), EPOFIT (INTAS PHARMA), ERYKINE (INTAS BIOPHARMACEUTICA), WEPOX (WOCKHARDT BIOTECH), ESPOGEN (LG LIFE SCIENCES), RELIPOIETIN (RELIANCE LIFE SCIENCES), SHANPOIETIN (SHANTHA BIOTECHNICS LTD), ZYROP (CADILA HEALTHCARE LTD.), EPIAO (RHUEPO) (SHENYANG SUNSHINE PHARMACEUTICAL CO. LTD), CINNAPOIETIN (CINNAGEN).

In some embodiments, when targeting EPO, the present invention relates to combination therapy with a blood transfusion. For instance, the present compositions may supplement a blood transfusion. In some embodiments, when targeting EPO, the present invention relates to combination therapy with iron supplements.

In some embodiments, the present invention also relates to the following protein targets, e.g. in the treatment of disease: growth hormone (GH) e.g. human and bovine growth hormone, growth hormone-releasing hormones; interferon including α-, β-, or γ-interferons, etc., interleukin-I; interleukin-II; erythropoietin including α- and β-erythropoietin (EPO), granulocyte colony stimulating factor (GCSF), granulocyte macrophage colony stimulating factor (GM-CSF), anti-agiogenic proteins (e.g., angiostatin, endostatin) PACAP polypeptide (pituitary adenylate cyclase activating polypeptide), vasoactive intestinal peptide (VIP), thyrotrophin releasing hormone (TRH), corticotropin releasing hormone (CRH), vasopressin, arginine vasopressin (AVP), angiotensin, calcitonin, atrial naturetic factor, somatostatin, adrenocorticotropin, gonadotropin releasing hormone, oxytocin, insulin, somatotropin, plasminogen tissue activator, coagulation factors including coagulation factors VIII and IX, glucosylceramidase, sargramostim, lenograstin, filgrastin, dornase-α, molgramostim, PEG-L-asparaginase, PEG-adenosine deaminase, hirudin, eptacog-α (human blood coagulation factor VIIa) nerve growth factors, transforming growth factor, epidermal growth factor, basic fibroblast growth factor, VEGF; heparin including low molecular weight heparin, calcitonin; antigens; monoclonal antibodies; vancomycin; desferrioxamine (DFO); parathyroid hormone, an immunogen or antigen, and an antibody such as a monoclonal antibody.

In some embodiments, the present methods allow for effective additional therapeutic agent (e.g. those described herein) activity and/or targeting to a cell and/or tissue of interest. For example, the present synthetic RNA can lead to increased expression of one or more targeting molecules that direct an additional therapeutic to the location of therapy. For example, the additional therapeutic agent may have a binding partner that the synthetic RNA encodes. For example, the synthetic RNA may induce the expression of an antigen that directs the therapeutic activity of an antibody that may be used in combination (e.g. herceptin, rituxan, campath, gemtuzumab, herceptin, panorex, rituximab, bexxar, edrecolomab, alemtuzumab, mylotrag, IMC-C225, smartin 195, and mitomomab). In some embodiments, the synthetic RNA can be injected directly into one or more of the tumors described herein and home the therapeutic antibody to the tumor.

In some embodiments, the present methods allow for effective additional therapeutic agent generation, especially when the additional therapeutic agent is a prodrug, for example, to produce an active form of the drug. In some embodiments, the synthetic RNA can be injected directly into one or more of the tumors described herein and home the prodrug to the tumor. For instance, the synthetic RNA may encode an enzyme that catalyzes the localized conversion of a non-toxic, systemically delivered agent into a potent chemotherapeutic agent. By way of illustration (note that any of the prodrugs or drugs listed herein are additional agents as used herein):

Enzyme Prodrug Drug aldehyde oxidases 5-Ethynyl-2(1H)-pyrimidinone 5-Ethynyluracil aldehyde oxidases IPdR IUdR aldehyde oxidases 5-FP 5-FU amino acid oxidases d-alanine Hydrogen peroxide amino acid oxidases SeCys conjugates Selenols and hydrogen peroxide cytochrome P450 reductase Menadione Semiquinone radical cytochrome P450 reductase Mitomycin C Quinone methide intermediate cytochrome P450 reductase Tirapazamine Nitroxide radical cytochrome P450 reductase EO9 Unidentified semiquinone radical DT-diaphorase Streptonigrin Unidentified DT-diaphorase Mitomycin C Quinone methide intermediate DT-diaphorase CB 1954 5-(Aziridin-1-yl)-4-hydroxyl-amino-2- nitrobenzamide DT-diaphorase Diaziquone Semiguinone radical^(a) cytochrome P450 Ipomeanol Unidentified (possibly furan epoxide) cytochrome P450 Ftorafur (tegafur) 5-FU cytochrome P450 Dacarbazine HHMTIC cytochrome P450 Trofosfamide Trofosfamide mustard cytochrome P450 Ifosfamide Isophosphamide mustard cytochrome P450 Cyclophosphamide Phosphoramide mustard cytochrome P450 AQ4N AQ4 tyrosinase 2,4-Dihydroxyphenylalanine 6-Hydroxydopa tyrosinase 4-S-CAP BQ tyrosinase GHB GBQ tyrosinase Substituted phenols Orthoquinones tyrosinase Phenyl mustards Phenol mustard tyrosinase Urea mustards Unidentified glutathione S-transferase TER286 Aziridinium agent glutathione S-transferase S-CPHC-ethylsulfoxide S-CPHC-glutathione glutathione S-transferase PTA 6-MP carboxylesterase CPT-11 SN-38 carboxylesterase Paclitaxel-2-ethylcarbonate Paclitaxel carboxylesterase Capecitabine 5′-Deoxy-5-fluorocytidine (5-FU) carboxylesterase Tertiary amidomethyl esters Carboxylic acids and amines^(a) alkaline phosphatase Amifostine WR-1065 alkaline phosphatase 3-AP phosphate 3-AP β-glucuronidase BHAMG pHAM β-glucuronidase Epirubicin-glucuronide Epirubicin β-glucuronidase HMR 1826 Doxorubicin β-glucuronidase DNR-GA3 Daunorubicin β-glucuronidase DOX-GA3 Doxorubicin β-glucuronidase Paclitaxel glucuronide Paclitaxel β-glucuronidase 5-FU glucuronide 5-FU cysteine conjugate β-lyase PC 6-MP cysteine conjugate β-lyase GC 6-Thioguanine cysteine conjugate β-lyase SeCys conjugate Selenol Nitroreductase CB 1954 5-(Aziridin-1-yl)-4- hydroxyl-amino-2- nitro- benzamide Cytochrome P450 4-Ipomeanol Unidentified (furan epoxide is speculated) Cytochrome P450 Ifosfamide Isophosphoramide mustard Cytochrome P450 Cyclophosphamide Phosphoramide mustard Purine-nucleoside Fludarabine 2-Fluoroadenine phosphorylase Purine-nucleoside MeP-dR MeP phosphorylase Thymidine kinase Ganciclovir Ganciclovir-triphosphate nucleotide Alkaline phosphatase Etoposide phosphate Etoposide Alkaline phosphatase Mitomycin C phosphate Mitomycin C Alkaline phosphatase POMP POM Alkaline phosphatase N-(4-phosphonooxy)- Doxorubicin phenylacetyl)doxorubicin β-Glucuronidase Glucuronidated Nornitrogen mustard Oxazolidinone β-Glucuronidase Glucuronidated 9-amino- 9-Aminocamptothecin camptothecin β-Glucuronidase Glucuronide mustard Mustard Carboxypeptidase Methotrexate-amino acids Methotrexate Carboxypeptidase CMDA Benzoic acid mustard Penicillin amidase DPO Doxorubicin Penicillin amidase MelPO Melphalan Penicillin amidase NHPAP Palytoxin Penicillin amidase N-(phenylacetyl) doxorubicin Doxorubicin Penicillin amidase N-(phenylacetyl) melphalan Melphalan β-Lactamase LY 266070 DAVLBHYD β-Lactamase C-DOX Doxorubicin β-Lactamase PRODOX Doxorubicin β-Lactamase CM Phenylenediamine mustard β-Lactamase CCM Phenylenediamine mustard β-Lactamase Cephalosporin-DACCP DACCP β-Lactamase PROTAX Taxol β-Lactamase Cephalosporin mitomycin C Mitomycin C β-Lactamase C-Mel Melphalan Cytosine deaminase 5-Fluorocytosine 5-Fluorouracil Methionine γ-lyase Selenomethionine Methylselenol Methionine γ-lyase Trifluoromethionine CSF₂

In certain embodiments, the synthetic RNA may encode an enzyme that catalyzes the conversion of 5-FU and/or Doxorubicin from various prodrugs, as illustrated by the examples below:

5-FU Prodrugs and Enzymes 5-FP Aldehyde oxidase Ftorafur P450 5′-DFUR Thymidine phosphorylase 5-FU glucuronide β-Glucuronidase 5-FC Cytosine deaminase Doxorubicin Prodrugs and Enzymes N-(4-phosphono-oxy)-phenylacetyl) Alkaline phosphatase doxorubicin HMR 1826 β-Glucuronidase DOX-GA3 β-Glucuronidase DPO Penicillin amidase N-(phenyacetyl) doxorubicin Penicillin amidase C-DOX β-Lactamase PRODOX β-Lactamase

In some embodiments, the nucleic acid drugs at the doses and regimens described herein may be used in combination with one or more additional agents (aka adjuvant therapy or combination agent). In some embodiments, the nucleic acid drugs at the doses and regimens described herein may be used in a human patient undergoing treatment with one or more additional agents. In some embodiments, the nucleic acid drug is used as an adjuvant or neoadjuvant to any of the additional agents described herein. In some embodiments, the invention pertains to co-administration and/or co-formulation. Any of the compositions described herein may be co-formulated and/or co-administered. In some embodiments, any nucleic acid drug described herein acts synergistically when co-administered with another agent and may be administered at doses that are lower than the doses commonly employed when such agents are used as monotherapy.

In some embodiments, any nucleic acid drug described herein may include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the composition such that covalent attachment does not prevent the activity of the composition. For example, but not by way of limitation, derivatives include composition that have been modified by, inter alia, glycosylation, lipidation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of turicamycin, etc. Additionally, the derivative can contain one or more non-classical amino acids. In various embodiments, one or more additional agents (aka adjuvant therapy or combination agent) may be conjugated to any nucleic acid drug described herein.

Contacting a cell with a steroid can suppress the innate immune response to foreign nucleic acids, and can increase the efficiency of nucleic acid delivery and translation. Certain embodiments are therefore directed to contacting a cell with a steroid. Other embodiments are directed to administering a steroid to a patient. Illustrative steroids include corticosteroid steroids. In some embodiments, the steroid is one or more of cortisone, hydrocortisone, prednisone, prednisolone, dexamethasone, triamcinolone, and betamethasone. In one embodiment, the steroid is hydrocortisone. In another embodiment, the steroid is dexamethasone.

Other embodiments are directed to administering to a patient a member of the group: an antibiotic, an antimycotic, and an RNAse inhibitor.

Botulinum toxin type A has been approved by the US Food and Drug Administration (FDA) for the treatment of essential blepharospasm, strabismus and hemifacial spasm in patients over the age of twelve, cervical dystonia, glabellar line (facial) wrinkles and for treating hyperhydrosis and botulinum toxin type B has been approved for the treatment of cervical dystonia. The present compositions may be combined with these toxins in the treatment of these diseases and related diseases. Some embodiments are directed to a nucleic acid drug targeting a neurotoxin. In various embodiments, the neurotoxin is a botulinum toxin or a biologically active fragment, variant, analogue or family-member thereof.

Further the combination of any one of the aforementioned toxins may be used in combination with the present compositions for various cosmetic procedures, including, without limitation, facial wrinkles, hyperkinetic skin lines, glabellar lines, crow's feet, marionette lines, skin disorders, nasolabial folds, blepharospasm, strabismus, hemifacial spasms and sweating disorders. Alternatively, the present compositions may be used to in these cosmetic procedures as a monotherapy.

Certain embodiments are directed to a combination therapy comprising one or more of the therapeutic or cosmetic compositions of the present invention and one or more adjuvant therapies or cosmetic treatments. In certain embodiments, one or more of the therapeutic or cosmetic compositions of the present invention are administered to a subject which is undergoing treatment with one or more adjuvant therapies or cosmetic treatments. Example adjuvant therapies and cosmetic treatments are set forth in Table 3 and Table 5 of U.S. Provisional Application No. 61/721,302, the contents of which are hereby incorporated by reference, and are given by way of example, and not by way of limitation.

TABLE 3 Illustrative Adjuvant Therapies Example Therapy/Treatment Class Disease/Condition Therapy/Treatment Acetylcholinesterase inhibitors Myasthenia gravis, Glaucoma, Edrophonium Alzheimer's disease, Lewy body dementia, Postural tachycardia syndrome Angiotensin-converting-enzyme Hypertension, Congestive heart failure Perindopril inhibitor Alkylating agents Cancer Cisplatin Angiogenesis inhibitors Cancer, Macular degeneration Bevacizumab Angiotensin II receptor antagonists Hypertension, Diabetic nephropathy, Valsartan Congestive heart failure Antibiotics Bacterial infection Amoxicillin Antidiabetic drugs Diabetes Metformin Antimetabolites Cancer, Infection 5-fluorouracil (5FU) Antisense oligonucleotides Cancer, Diabetes, Amyotrophic lateral Mipomersen sclerosis (ALS), Hypercholesterolemia Cytotoxic antibiotics Cancer Doxorubicin Deep-brain stimulation Chronic pain, Parkinson's disease, N/A Tremor, Dystonia Dopamine agonists Parkinson's disease, Type II diabetes, Bromocriptine Pituitary tumors Entry/Fusion inhibitors HIV/AIDS Maraviroc Glucagon-like peptide-1 agonists Diabetes Exenatide Glucocorticoids Asthma, Adrenal insufficiency, Dexamethasone Inflammatory diseases, Immune diseases, Bacterial meningitis Immunosuppressive drugs Organ transplantation, Inflammatory Azathioprine diseases, Immune diseases Insulin/Insulin analogs Diabetes NPH insulin Integrase inhibitors HIV/AIDS Raltegravir MAO-B inhibitors Parkinson's disease, Depression, Selegiline Dementia Maturation inhibitors HIV/AIDS Bevirimat Nucleoside analog reverse- HIV/AIDS, Hepatitis B Lamivudine transcriptase inhibitors Nucleotide analog reverse- HIV/AIDS, Hepatitis B Tenofovir transcriptase inhibitors Non-nucleoside reverse-transcriptase HIV/AIDS Rilpivirine inhibitors Pegylated interferon Hepatitis B/C, Multiple sclerosis Interferon beta-1a Plant alkaloids/terpenoids Cancer Paclitaxel Protease inhibitors HIV/AIDS, Hepatitis C, Other viral Telaprevir infections Radiotherapy Cancer Brachytherapy Renin inhibitors Hypertension Aliskiren Statins Hypercholesterolemia Atorvastatin Topoisomerase inhibitors Cancer Topotecan Vasopressin receptor antagonist Hyponatremia, Kidney disease Tolvaptan Dermal filler Wrinkles, aged skin Hyaluronic Acid Botulinum toxin Wrinkles, aged skin botulinum toxin type A Induction of skin repair Acne scars, aged skin Laser treatment, dermabrasion

In some embodiments, the additional agent is a cytotoxic agent, comprising, in illustrative embodiments, a toxin, a chemotherapeutic agent, a radioisotope, and an agent that causes apoptosis or cell death.

Illustrative cytotoxic agents include, but are not limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine; alkylating agents such as mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU), mitomycin C, lomustine (CCNU), 1-methylnitrosourea, cyclothosphamide, mechlorethamine, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP) cisplatin and carboplatin (paraplatin); anthracyclines include daunorubicin (formerly daunomycin), doxorubicin (adriamycin), detorubicin, carminomycin, idarubicin, epirubicin, mitoxantrone and bisantrene; antibiotics include dactinomycin (actinomycin D), bleomycin, calicheamicin, mithramycin, and anthramycin (AMC); and antimytotic agents such as the vinca alkaloids, vincristine and vinblastine. Other cytotoxic agents include paclitaxel (taxol), ricin, pseudomonas exotoxin, gemcitabine, cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, tenoposide, colchicin, dihydroxy anthracin dione, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mytotane (O,P′-(DDD)), interferons, and mixtures of these cytotoxic agents.

Further cytotoxic agents include, but are not limited to, chemotherapeutic agents such as carboplatin, cisplatin, paclitaxel, gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin C, actinomycin D, cyclophosphamide, vincristine, bleomycin, VEGF antagonists, EGFR antagonists, platins, taxols, irinotecan, 5-fluorouracil, gemcytabine, leucovorine, steroids, cyclophosphamide, melphalan, vinca alkaloids (e.g., vinblastine, vincristine, vindesine and vinorelbine), mustines, tyrosine kinase inhibitors, radiotherapy, sex hormone antagonists, selective androgen receptor modulators, selective estrogen receptor modulators, PDGF antagonists, TNF antagonists, IL-1 antagonists, interleukins (e.g. IL-12 or IL-2), IL-12R antagonists, Toxin conjugated monoclonal antibodies, tumor antigen specific monoclonal antibodies, Erbitux, Avastin, Pertuzumab, anti-CD20 antibodies, Rituxan, ocrelizumab, ofatumumab, DXL625, HERCEPTIN, or any combination thereof. Toxic enzymes from plants and bacteria such as ricin, diphtheria toxin and Pseudomonas toxin may be conjugated to the therapeutic agents (e.g. antibodies) to generate cell-type-specific-killing reagents (Youle, et al., Proc. Nat'l Acad. Sci. USA 77:5483 (1980); Gilliland, et al., Proc. Nat'l Acad. Sci. USA 77:4539 (1980); Krolick, et al., Proc. Nat'l Acad. Sci. USA 77:5419 (1980)).

Other cytotoxic agents include cytotoxic ribonucleases as described by Goldenberg in U.S. Pat. No. 6,653,104. Embodiments of the invention also relate to radioimmunoconjugates where a radionuclide that emits alpha or beta particles is stably coupled to the antibody, or binding fragments thereof, with or without the use of a complex-forming agent. Such radionuclides include beta-emitters such as Phosphorus-32, Scandium-47, Copper-67, Gallium-67, Yttrium-88, Yttrium-90, Iodine-125, Iodine-131, Samarium-153, Lutetium-177, Rhenium-186 or Rhenium-188, and alpha-emitters such as Astatine-211, Lead-212, Bismuth-212, Bismuth-213 or Actinium-225.

Illustrative detectable moieties further include, but are not limited to, horseradish peroxidase, acetylcholinesterase, alkaline phosphatase, beta-galactosidase and luciferase. Further exemplary fluorescent materials include, but are not limited to, rhodamine, fluorescein, fluorescein isothiocyanate, umbelliferone, dichlorotriazinylamine, phycoerythrin and dansyl chloride. Further exemplary chemiluminescent moieties include, but are not limited to, luminol. Further exemplary bioluminescent materials include, but are not limited to, luciferin and aequorin. Further exemplary radioactive materials include, but are not limited to, Iodine-125, Carbon-14, Sulfur-35, Tritium and Phosphorus-32.

The dosage of any additional agent described herein as well as the dosing schedule can depend on various parameters, including, but not limited to, the human patient's general health, and the administering physician's and/or human patient's discretion. Co-administration may be simultaneous or sequential. Any additional agent described herein, can be administered prior to (e.g., about 5 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, about 96 hours, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, 8 weeks, or about 12 weeks before), concurrently with, or subsequent to (e.g., about 5 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, about 96 hours, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 8 weeks, or about 12 weeks after) the administration of the nucleic acid drug to a human patient in need thereof. In various embodiments any agent described herein is administered about 1 minute apart, about 10 minutes apart, about 30 minutes apart, less than about 1 hour apart, about 1 hour apart, about 1 hour to about 2 hours apart, about 2 hours to about 3 hours apart, about 3 hours to about 4 hours apart, about 4 hours to about 5 hours apart, about 5 hours to about 6 hours apart, about 6 hours to about 7 hours apart, about 7 hours to about 8 hours apart, about 8 hours to about 9 hours apart, about 9 hours to about 10 hours apart, about 10 hours to about 11 hours apart, about 11 hours to about 12 hours apart, no more than about 24 hours apart or no more than about 48 hours apart.

In a specific embodiment, the combination regimen is designed to exploit the finding that the nucleic acid drug dosing and formulation of the present invention has potent effects quickly (e.g. in about 6, or about 12, or about 24, or about 30, or about 36, or about 48 hours) and the effects can last about 7 days, or longer.

The dose of a nucleic acid drug is disclosed herein. In general, the dose of any additional agent that is useful is known to those in the art. For example, doses may be determined with reference Physicians' Desk Reference, 66th Edition, PDR Network; 2012 Edition (Dec. 27, 2011), the contents of which are incorporated by reference in its entirety. In some embodiments, the present invention allows a patient to receive doses that exceed those determined with reference Physicians' Desk Reference. The dosage of any additional agent described herein can depend on several factors including the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the human patient to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular human patient may affect dosage used. Furthermore, the exact individual dosages can be adjusted somewhat depending on a variety of factors, including the specific combination of the agents being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the particular disease being treated, the severity of the disorder, and the anatomical location of the disorder. Some variations in the dosage can be expected.

Cells, tissues, organs, and organisms, including, but not limited to, humans, have several characteristics that can inhibit or prevent the delivery of nucleic acids, including, for example, the stratum corneum, which can serve as a barrier to foreign organisms and nucleic acids. These characteristics can thus inhibit the effects of therapeutics and cosmetics comprising nucleic acids. It has now been discovered that many of these characteristics can be circumvented or overcome using a patch comprising a flexible membrane and a plurality of needles, and that such a patch can serve as an effective and safe article for the delivery of nucleic acids. Certain embodiments are therefore directed to a nucleic acid delivery patch. In one embodiment, the nucleic acid delivery patch comprises a flexible membrane. In another embodiment, the nucleic acid delivery patch comprises a plurality of needles. In yet another embodiment, the plurality of needles are attached to the flexible membrane. In some embodiments, the patch comprises a nucleic acid. In one embodiment, the nucleic acid is present in solution. In one embodiment, the plurality of needles include one or more needles having a lumen. In another embodiment, the patch further comprises a second flexible membrane. In yet another embodiment, the flexible membrane and the second flexible membrane are arranged to form a cavity. In a further embodiment, the cavity contains a nucleic acid. In a still further embodiment, the membrane comprises one or more holes through which a nucleic acid can pass. In a still further embodiment, one or more holes and one or more needles having a lumen are arranged to allow the passage of a solution containing a nucleic acid through at least one of the one or more holes and through at least one of the one or more needles having a lumen. In some embodiments, the patch is configured to deliver a solution to the skin. In one embodiment, the solution comprises a nucleic acid. In another embodiment, the solution comprises a vehicle. In yet another embodiment, the vehicle is a lipid or lipidoid. In a still further embodiment, the vehicle is a lipid-based transfection reagent.

The cell membrane can serve as a barrier to foreign nucleic acids. It has now been discovered that combining the patch of the present invention with an electric field can increase the efficiency of nucleic acid delivery. Certain embodiments are therefore directed to a nucleic acid delivery patch comprising a plurality of needles, wherein at least two needles form part of a high-voltage circuit. Certain embodiments are directed to an implantable “tattoo” for microneedle delivery (see, e.g. Nature Materials 12, pp 367-376 (2013), the contents of which are hereby incorporated by reference in their entirety). In one embodiment, the high-voltage circuit generates a voltage greater than about 10V. In another embodiment, the high-voltage circuit generates a voltage greater than about 20V. In yet another embodiment, an electric field is produced between two of the needles. In a further embodiment, the magnitude of the electric field is at least about 100V/cm. In a still further embodiment, the magnitude of the electric field is at least about 200V/cm. In some embodiments, the patch is configured to deliver a nucleic acid to the epidermis. In other embodiments, the patch is configured to deliver a nucleic acid to the dermis. In still other embodiments, the patch is configured to deliver a nucleic acid to sub-dermal tissue. In still other embodiments, the patch is configured to deliver a nucleic acid to muscle. Certain embodiments are directed to a nucleic acid delivery patch comprising a plurality of electrodes. In one embodiment, the plurality of electrodes is attached to a flexible membrane. Other embodiments are directed to a nucleic acid delivery patch comprising a rigid structure. In one embodiment, a plurality of electrodes are attached to the rigid structure.

In some embodiments, the compositions described herein are administered using an array of needles covering an affected area of the subject. In some embodiments, the treatment area is mechanically massaged after administration. In some embodiments, the treatment area is exposed to electric pulses after administration. In some embodiments, the electric pulses are between about 10V and about 200V for from about 50 microseconds to about 1 second. In some embodiments, the electric pulses are generated around the treatment area by a multielectrode array.

In some embodiments, the present invention provides a patch delivery system, comprising a non-viral RNA transfection composition enclosed within a membrane, and an array of delivery needles delivering from about 10 ng to about 2000 ng of RNA per treatment area of about 100 cm² or less, or about 50 cm² or less, or about 10 cm² or less, or about 5 cm² or less, or about 1 cm² or less, or about 0.5 cm² or less, or about 0.2 cm² or less. In some embodiments, the non-viral transfection composition contains from about 10 ng to about 2000 ng per injection volume of about 20 μL to about 1 ml. In some embodiments, each needle delivers an injection volume of between 1 μL and 500 μL.

In some embodiments, the delivery patch comprises an acrylic reservoir that holds the nucleic acid drug. In some embodiments, a silicon adhesive is added to create a semisolid suspension of microscopic, concentrated drug cells. Further, some embodiments pride a patch that is associated with one or more enhancers (these include, without limitation, iontophoresis, ultrasound, chemicals including gels, microneedles, sonophoresis, lasers, and electroporatic methods).

In some embodiments, the delivery is effected via a gel, optionally a hydro alcoholic gel containing a combination of enhancers (e.g. COMBIGEL (ANTARES PHARMA)).

In various embodiments, the RNA is delivered using needle arrays. Illustrative needle arrays include, but are not limited to AdminPen 600 and those described in U.S. Pat. Nos. 7,658,728, 7,785,301, and 8,414,548, the entire disclosure of which are hereby incorporated by reference. Other examples of needles include, for example, the 3M™ Hollow Microstructured Transdermal System and the 3M Solid Microstructured Transdermal Systems (sMTS). See, e.g. U.S. Pat. Nos. 3,034,507 and 3,675,766; Microneedles for Transdermal Drug Delivery. Advanced Drug Delivery Reviews. 56: 581-587 (2004); Pharm Res. 2011 January; 28(1): 31-40, the entire contents of which are hereby incorporated by reference in their entireties.

In some embodiments, microneedles and/or microneedle arrays may be used. In various embodiments, the microneedles and/or microneedle arrays may be, without limitation, solid, RNA-coated, dissolving, biodegradable, and/or hollow. In some embodiments, the delivery is effected via a microneedle system, optionally combined with an electronically controlled micropump that delivers the drug at specific times or upon demand. For example, the MACROFLUX (Alza) system may be used.

Other embodiments are directed to a method for delivering a nucleic acid to a cell in vivo comprising applying a nucleic acid to a tissue containing a cell in vivo. In one embodiment, the method further comprises applying a transient electric field in the vicinity of the cell. In another embodiment, the method results in the cell in vivo internalizing the nucleic acid. In yet another embodiment, the nucleic acid comprises synthetic RNA. In a further embodiment, the method further results in the cell internalizing a therapeutically or cosmetically effective amount of the nucleic acid. In one embodiment, the cell is a skin cell. In another embodiment, the cell is a muscle cell. In yet another embodiment, the cell is a dermal fibroblast. In a further embodiment, the cell is a keratinocyte. In a still further embodiment, the cell is a myoblast. In some embodiments, the nucleic acid comprises a protein of interest. In one embodiment, the protein of interest is a fluorescent protein. In another embodiment, the protein of interest is an extracellular-matrix protein. In yet another embodiment, the protein of interest is a member of the group: elastin, collagen, laminin, fibronectin, vitronectin, lysyl oxidase, elastin binding protein, a growth factor, fibroblast growth factor, transforming growth factor beta, granulocyte colony-stimulating factor, a matrix metalloproteinase, an actin, fibrillin, microfibril-associated glycoprotein, a lysyl-oxidase-like protein, platelet-derived growth factor, a lipase, an uncoupling protein, thermogenin, filaggrin, a fibroblast growth factor, an antibody, and a protein involved with pigment production. In some embodiments, the method further comprises delivering the nucleic acid to the epidermis. In other embodiments, the method further comprises delivering the nucleic acid to the dermis. In still other embodiments, the method further comprises delivering the nucleic acid below the dermis. In one embodiment, the delivering is by injection. In another embodiment, the delivering is by injection using a micro-needle array. In yet another embodiment, the delivering is by topical administration. In a further embodiment, the delivering comprises disruption or removal of a part of the tissue. In a still further embodiment, the delivering comprises disruption or removal of the stratum corneum. In some embodiments, the nucleic acid is present in solution. In one embodiment, the solution comprises a growth factor. In another embodiment, the growth factor is a member of the group: a fibroblast growth factor and a transforming growth factor. In yet another embodiment, the growth factor is a member of the group: basis fibroblast growth factor and transforming growth factor beta. In other embodiments, the solution comprises cholesterol.

In another embodiment, the method further comprises contacting the cell with one or more nucleic acid molecules. In yet another embodiment, at least one of the one or more nucleic acid molecules encodes a protein of interest. In a further embodiment, the method results in the cell expressing the protein of interest. In a still further embodiment, the method results in the cell expressing a therapeutically or cosmetically effective amount of the protein of interest.

In another embodiment, the cell is contacted with a nucleic acid molecule. In yet another embodiment, the method results in the cell internalizing the nucleic acid molecule. In a further embodiment, the method results in the cell internalizing a therapeutically or cosmetically effective amount of the nucleic acid molecule. In one embodiment, the nucleic acid encodes a protein of interest. In one embodiment, the nucleic acid molecule comprises a member of the group: a dsDNA molecule, a ssDNA molecule, a RNA molecule, a dsRNA molecule, a ssRNA molecule, a plasmid, an oligonucleotide, a synthetic RNA molecule, a miRNA molecule, an mRNA molecule, and an siRNA molecule. In various embodiments, the RNA comprises one or more non-canonical nucleotides.

In some embodiments, the present invention relates to one or more administration techniques described in U.S. Pat. Nos. 5,711,964; 5,891,468; 6,316,260; 6,413,544; 6,770,291; and 7,390,780, the entire contents of which are hereby incorporated by reference in their entireties.

The invention also provides kits that can simplify the administration of the nucleic acid drugs described herein and/or any additional agent described herein. An illustrative kit of the invention comprises a nucleic acid drug and/or any additional agent described herein in unit dosage form. In one embodiment, the unit dosage form is a container, such as a pre-filled syringe, which can be sterile, containing any agent described herein and a pharmaceutically acceptable carrier, diluent, excipient, or vehicle. The kit can further comprise a label or printed instructions instructing the use of any agent described herein. The kit or one or more components of the kit may be stored at room temperature, about 4° C., about −20° C., about −80° C., or about −196° C. The kit may also include a lid speculum, topical anesthetic, and a cleaning agent for the administration location. The kit can also further comprise one or more additional agent described herein. In one embodiment, the kit comprises a container containing an effective amount of a nucleic acid drug as disclosed herein and an effective amount of another composition, such as an additional agent as described herein. In some embodiments, the unit dosage form is a pre-loaded (a.k.a. pre-dosed or pre-filled) syringe or a pen needle injector (injection pen)). Such unit dosage forms may comprise the effective doses of nucleic acid drug described herein, e.g. about 10 ng to about 2000 ng, e.g. about 10 ng, or about 20 ng, or about 50 ng, or about 100 ng, or about 200 ng, or about 300 ng, or about 400 ng, or about 500 ng, or about 600 ng, or about 700 ng, or about 800 ng, or about 900 ng, or about 1000 ng, or about 1100 ng, or about 1200 ng, or about 1300 ng, or about 1400 ng, or about 1500 ng, or about 1600 ng, or about 1700 ng, or about 1800 ng, or about 1900 ng, or about 2000 ng, or about 3000 ng, or about 4000 ng, or about 5000 ng.

Some embodiments are directed to synthetic RNA molecules with low toxicity and high translation efficiency. Other embodiments are directed to a cell-culture medium for high-efficiency in vivo transfection, reprogramming, and gene editing of cells. Other embodiments pertain to methods for producing synthetic RNA molecules encoding reprogramming proteins. Still further embodiments pertain to methods for producing synthetic RNA molecules encoding gene-editing proteins.

Some embodiments are directed to methods of gene-editing and/or gene correction. Some embodiments encompass synthetic RNA-based gene-editing and/or gene correction, e.g. with RNA comprising non-canonical nucleotides, e.g. RNA encoding one or more of a nuclease, a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease, a meganuclease, a nickase, a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein a DNA-repair protein, a DNA-modification protein, a base-modification protein, a DNA methyltransferase, an protein that causes DNA demethylation, an enzyme for which DNA is a substrate or a natural or engineered variant, family-member, orthologue, fragment or fusion construct thereof. In some embodiments, the efficiency of the gene-editing and/or gene correction is high, for example, higher than DNA-based gene editing and/or gene correction. In some embodiments, the present methods of gene-editing and/or gene correction are efficient enough for in vivo application. In some embodiments, the present methods of gene-editing and/or gene correction are efficient enough to not require cellular selection (e.g. selection of cells that have been edited). In various embodiments, the efficiency of gene-editing of the present methods is about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 100%. In various embodiments, the efficiency of gene-correction of the present methods is about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 100%

Some embodiments are directed to high-efficiency gene-editing proteins comprising engineered nuclease cleavage or DNA-modification domains. Other embodiments are directed to high-fidelity gene-editing proteins comprising engineered nuclease cleavage or DNA-modification domains. Various embodiments are directed to high-efficiency gene-editing proteins comprising engineered DNA-binding domains. Other embodiments are directed to high-fidelity gene-editing proteins comprising engineered DNA-binding domains. Still other embodiments are directed to gene-editing proteins comprising engineered repeat sequences. Some embodiments are directed to gene-editing proteins comprising one or more CRISPR associated family members. Some embodiments are directed to methods for altering the DNA sequence of a cell by transfecting the cell with or inducing the cell to express a gene-editing protein. Other embodiments are directed to methods for altering the DNA sequence of a cell that is present in an in vitro culture. Still further embodiments are directed to methods for altering the DNA sequence of a cell that is present in vivo.

Some embodiments are directed to methods of modulating the secretion or subcellular localization of a polypeptide. In such embodiments, the present invention provides an RNA encoding a protein comprising a signal peptide operably linked to a polypeptide having biological functionality. In an embodiment, the signal peptide modulates the secretion of the polypeptide. In another embodiment, the signal peptide modulates the subcellular localization of the polypeptide. For example, BMP7 comprising at least one FGF21 signal peptide may result in increased secretion of BMP7. In another example, BMP7 comprising at least one FGF21 signal peptide in place of the endogenous BMP7 signal peptide may result in increased secretion of BMP7. In various embodiments, any of the proteins listed in Table 2A or Table 2B comprising at least one signal peptide listed in the table below may result in increased secretion of the proteins.

SEQ ID Protein Signal Peptide NO Gaussia luciferase MGVKVLFALICIAVAEA 596 Human BMP7 MHVRSLRAAAPHSFVAL 597 WAPLFLLRSALA Human  MAFLWLLSCWALLGTTFG 598 chymotrypsinogen B Human  MLGITVLAALLACASS 599 chymotrypsinogen C Human EPO MGVHECPAWLWLLLSLL 600 SLPLGLPVLG Human FGF19 MRSGCVVVHVWILAGLW 601 LAVAGRP Human FGF21 MDSDETGFEHSGLWVSV 602 LAGLLLGACQA Human FGF23 MLGARLRLWVCALCSVC 603 SMSVLRA Human IL2 MYRMQLLSCIALSLALVTNS 604 Human IL22 MAALQKSVSSFLMGTLA 605 TSCLLLLALLVQGGAA Human IL6 MNSFSTSAFGPVAFSLG 606 LLLVLPAAFPAP Human Interferon  MALTFALLVALLVLSCK 607 Alpha-2 SSCSVG Human Interferon  MTNKCLLQIALLLCFST 608 Beta TALS Human Interferon  MKYTSYILAFQLCIVLG 609 Gamma SLGCYC Human Trypsin-1 MNPLLILTFVAAALA 610

Some embodiments are directed to methods of modulating the serum half-life, secretion, bioavailability, and/or activity of a protein comprising administering a RNA encoding the protein and a RNA encoding a receptor for the protein. For example, IL15 may be administered with its receptor, e.g., IL15RA to enhance one or more of the serum half-life, secretion, bioavailability, and activity of IL15.

Many proteins and peptides, when translated from in vitro transcribed RNA, can exhibit reduced activity resulting from incomplete or inadequate post-translational processing. It has now been discovered that the amount of active protein, polypeptide or peptide produced following RNA transfection can be increased by contacting cells with and/or inducing cells to express a second protein that is capable of processing a polypeptide into the protein, peptide or polypeptide. Certain embodiments are therefore directed to a method for inducing a cell to express an active protein, polypeptide or peptide comprising contacting the cell with a synthetic RNA molecule encoding a first polypeptide and contacting the cell with a second protein that is capable of processing the first polypeptide into an active protein, polypeptide or peptide.

In certain embodiments, the second protein is administered to a patient. In one embodiment, the second protein is a recombinant protein. In another embodiment, the cells are contacted with a synthetic RNA molecule encoding the second protein. In a further embodiment, the cells are contacted with and/or induced to express an inhibitor of a molecule that inhibits the second protein. In one embodiment, the inhibitor is a short interfering RNA molecule.

In certain embodiments, the second protein is a member of the PCSK family. In other embodiments, the second protein is a proprotein convertase. In still other embodiments, the second protein is a prohormone convertase. In still other embodiments, the second protein is a carboxypeptidase. In one embodiment, the second protein is PCSK3 (Furin/PACE). In another embodiment, the second protein is primarily secreted. In yet another embodiment, the second protein is primarily intracellular. In a further embodiment, the first polypeptide, protein, polypeptide or peptide is selected from the group: a product of proopiomelanocortin, renin, a product of enkephalin, a product of prodynorphin, somatostatin, insulin, agouti-related peptide, glucagon, parathyroid hormone, a member of the transforming growth factor beta superfamily, albumin, beta-secretase 1, nerve growth factor, caldesmon, the alpha-integrins, factor IX, α-melanocyte-stimulating hormone, adrenocorticotropic hormone, β-endorphin, and met-enkefalin.

Glycation and glycosylation are processes by which one or more sugar molecules are bound to a protein. It has now been discovered that altering the number or location of glycation and glycosylation sites can increase or decrease the stability of a protein. Certain embodiments are therefore directed to a protein with one or more glycation or glycosylation sites. In one embodiment, the protein is engineered to have more glycation or glycosylation sites than a natural variant of the protein. In another embodiment, the protein is engineered to have fewer glycation or glycosylation sites than a natural variant of the protein. In yet another embodiment, the protein has increased stability. In yet another embodiment, the protein has decreased stability. In some embodiments, the protein is a circulating protein. In one embodiment, the protein is erythropoietin or a biologically active fragment, variant, analogue, or family-member thereof. In another embodiment, the protein is darbepoetin alfa or a biologically active fragment, variant, analogue, or family-member thereof. In another embodiment, the protein is NOVEPOETIN or a biologically active fragment, variant, analogue, or family-member thereof.

It has been further discovered that in certain situations, including one or more steroids and/or one or more antioxidants in the transfection medium can increase in vivo transfection efficiency, in vivo reprogramming efficiency, and in vivo gene-editing efficiency. Certain embodiments are therefore directed to contacting a cell or patient with a glucocorticoid, such as hydrocortisone, prednisone, prednisolone, methylprednisolone, dexamethasone or betamethasone. Other embodiments are directed to a method for inducing a cell to express a protein of interest by contacting a cell with a medium containing a steroid and contacting the cell with one or more nucleic acid molecules. In one embodiment, the nucleic acid molecule comprises synthetic RNA. In another embodiment, the steroid is hydrocortisone. In yet another embodiment, the hydrocortisone is present in the medium at a concentration of between about 0.1 uM and about 10 uM, or about 1 uM. Other embodiments are directed to a method for inducing a cell in vivo to express a protein of interest by contacting the cell with a medium containing an antioxidant and contacting the cell with one or more nucleic acid molecules. In one embodiment, the antioxidant is ascorbic acid or ascorbic-acid-2-phosphate. In another embodiment, the ascorbic acid or ascorbic-acid-2-phosphate is present in the medium at a concentration of between about 0.5 mg/L and about 500 mg/L, including about 50 mg/L. Still other embodiments are directed to a method for reprogramming and/or gene-editing a cell in vivo by contacting the cell with a medium containing a steroid and/or an antioxidant and contacting the cell with one or more nucleic acid molecules, wherein the one or more nucleic acid molecules encodes one or more reprogramming and/or gene-editing proteins. In certain embodiments, the cell is present in an organism, and the steroid and/or antioxidant are delivered to the organism.

Adding transferrin to the complexation medium has been reported to increase the efficiency of plasmid transfection in certain situations. It has now been discovered that adding transferrin to the complexation medium can also increase the efficiency of in vivo transfection with synthetic RNA molecules. Certain embodiments are therefore directed to a method for inducing a cell in vivo to express a protein of interest by adding one or more synthetic RNA molecules and a transfection reagent to a solution containing transferrin. In one embodiment, the transferrin is present in the solution at a concentration of between about 1 mg/L and about 100 mg/L, such as about 5 mg/L. In another embodiment, the transferrin is recombinant.

In certain situations, including pertaining to culturing, it may be desirable to replace animal-derived components with non-animal-derived and/or recombinant components, in part because non-animal-derived and/or recombinant components can be produced with a higher degree of consistency than animal-derived components, and in part because non-animal-derived and/or recombinant components carry less risk of contamination with toxic and/or pathogenic substances than do animal-derived components. Certain embodiments are therefore directed to a protein that is non-animal-derived and/or recombinant. Other embodiments are directed to a medium, wherein some or all of the components of the medium are non-animal-derived and/or recombinant.

Other embodiments are directed to a method for transfecting a cell in vivo. In one embodiment, a cell in vivo is transfected with one or more nucleic acids, and the transfection is performed using a transfection reagent, such as a lipid-based transfection reagent. In one embodiment, the one or more nucleic acids includes at least one RNA molecule. In another embodiment, the cell is transfected with one or more nucleic acids, and the one or more nucleic acids encodes at least one of: p53, TERT, an antibody, an extracellular matrix protein, a cytokine, a secreted protein, a membrane-bound protein, an enzyme, a gene-editing protein, a chromatin-modifying protein, a DNA-binding protein, a transcription factor, a histone deacetylase, a pathogen-associated molecular pattern, and a tumor-associated antigen or a biologically active fragment, analogue, variant or family-member thereof. In another embodiment, the cell is transfected repeatedly, such as at least about 2 times during about 10 consecutive days, or at least about 3 times during about 7 consecutive days, or at least about 4 times during about 6 consecutive days. Some embodiments are directed to a method for increasing expression of telomerase in one of a fibroblast, a hematopoietic stem cell, a mesenchymal stem cells, a cardiac stem cell, a hair follicle stem cell, a neural stem cell, an intestinal stem cell, an endothelial stem cell, an olfactory stem cell, a neural crest stem cell, a testicular cell, and a keratinocyte. Some embodiments are directed to a method for increasing the length of telomeres in one of a fibroblast, a hematopoietic stem cell, a mesenchymal stem cells, a cardiac stem cell, a hair follicle stem cell, a neural stem cell, an intestinal stem cell, an endothelial stem cell, an olfactory stem cell, a neural crest stem cell, a testicular cell, and a keratinocyte. Other embodiments are directed to a method for isolating a cell from a patient, contacting the cell with a nucleic acid drug encoding a component of telomerase (e.g., TERT), and reintroducing the cell to the patient. Various embodiments are directed to a method for increasing the replicative potential of a cell.

Reprogramming can be performed by transfecting cells with one or more nucleic acids encoding one or more reprogramming factors. Examples of reprogramming factors include, but are not limited to: Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, I-Myc protein, TERT protein, Nanog protein, Lin28 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof. Certain embodiments are therefore directed to a method for reprogramming a cell in vivo. In one embodiment, the cell in vivo is reprogrammed by transfecting the cell with one or more nucleic acids encoding one or more reprogramming factors. In one embodiment, the one or more nucleic acids includes an RNA molecule that encodes Oct4 protein. In another embodiment, the one or more nucleic acids also includes one or more RNA molecules that encodes Sox2 protein, Klf4 protein, and c-Myc protein. In yet another embodiment, the one or more nucleic acids also includes an RNA molecule that encodes Lin28 protein. In one embodiment, the cell is a human skin cell, and the human skin cell is reprogrammed to a pluripotent stem cell. In another embodiment, the cell is a human skin cell, and the human skin cell is reprogrammed to a glucose-responsive insulin-producing cell. Examples of other cells that can be reprogrammed and other cells to which a cell can be reprogrammed include, but are not limited to: skin cells, pluripotent stem cells, mesenchymal stem cells, β-cells, retinal pigmented epithelial cells, hematopoietic cells, cardiac cells, airway epithelial cells, neural stem cells, neurons, glial cells, bone cells, blood cells, and dental pulp stem cells. In one embodiment, the cell is contacted with a medium that supports the reprogrammed cell. In one embodiment, the medium also supports the cell.

Importantly, infecting skin cells with viruses encoding Oct4, Sox2, Klf4, and c-Myc, combined with culturing the cells in a medium that supports the growth of cardiomyocytes, has been reported to cause reprogramming of the skin cells to cardiomyocytes, without first reprogramming the skin cells to pluripotent stem cells (See Efs et al Nat Cell Biol. 2011; 13:215-22, the contents of which are hereby incorporated by reference). In certain situations, direct reprogramming (reprogramming one somatic cell to another somatic cell without first reprogramming the somatic cell to a pluripotent stem cell, also known as “transdifferentiation”) may be desirable, in part because culturing pluripotent stem cells can be time-consuming and expensive, the additional handling involved in establishing and characterizing a stable pluripotent stem cell line can carry an increased risk of contamination, and the additional time in culture associated with first producing pluripotent stem cells can carry an increased risk of genomic instability and the acquisition of mutations, including point mutations, copy-number variations, and karyotypic abnormalities. Certain embodiments are therefore directed to a method for reprogramming a somatic cell in vivo, wherein the cell is reprogrammed to a somatic cell, and wherein a characterized pluripotent stem-cell line is not produced.

It has been further discovered that, in certain situations, fewer total transfections may be required to reprogram a cell according to the methods of the present invention than according to other methods. Certain embodiments are therefore directed to a method for reprogramming a cell in vivo, wherein between about 1 and about 12 transfections are performed during about 20 consecutive days, or between about 4 and about 10 transfections are performed during about 15 consecutive days, or between about 4 and about 8 transfections are performed during about 10 consecutive days. It is recognized that when a cell is contacted with a medium containing nucleic acid molecules, the cell may likely come into contact with and/or internalize more than one nucleic acid molecule either simultaneously or at different times. A cell can therefore be contacted with a nucleic acid more than once, e.g. repeatedly, even when a cell is contacted only once with a medium containing nucleic acids.

Of note, nucleic acids can contain one or more non-canonical or “modified” residues as described herein. For instance, any of the non-canonical nucleotides described herein can be used in the present reprogramming methods. In one embodiment, pseudouridine-5′-triphosphate can be substituted for uridine-5′-triphosphate in an in vitro-transcription reaction to yield synthetic RNA, wherein up to 100% of the uridine residues of the synthetic RNA may be replaced with pseudouridine residues. In vitro-transcription can yield RNA with residual immunogenicity, even when pseudouridine and 5-methylcytidine are completely substituted for uridine and cytidine, respectively (see, e.g., Angel. Reprogramming Human Somatic Cells to Pluripotency Using RNA [Doctoral Thesis]. Cambridge, Mass.: MIT; 2011, the contents of which are hereby incorporated by reference). For this reason, it is common to add an immunosuppressant to the transfection medium when transfecting cells with RNA. In certain situations, adding an immunosuppressant to the transfection medium may not be desirable, in part because the recombinant immunosuppressant most commonly used for this purpose, B18R, can be expensive and difficult to manufacture. It has now been discovered that cells in vivo can be transfected and/or reprogrammed according to the methods of the present invention, without using B18R or any other immunosuppressant. It has been further discovered that reprogramming cells in vivo according to the methods of the present invention without using immunosuppressants can be rapid, efficient, and reliable. Certain embodiments are therefore directed to a method for transfecting a cell in vivo, wherein the transfection medium does not contain an immunosuppressant. Other embodiments are directed to a method for reprogramming a cell in vivo, wherein the transfection medium does not contain an immunosuppressant. In certain situations, for example when using a high cell density, it may be beneficial to add an immunosuppressant to the transfection medium. Certain embodiments are therefore directed to a method for transfecting a cell in vivo, wherein the transfection medium contains an immunosuppressant. Other embodiments are directed to a method for reprogramming a cell in vivo, wherein the transfection medium contains an immunosuppressant. In one embodiment, the immunosuppressant is B18R or a biologically active fragment, analogue, variant or family-member thereof or dexamethasone or a derivative thereof. In one embodiment, the transfection medium does not contain an immunosuppressant, and the nucleic-acid dose is chosen to prevent excessive toxicity. In another embodiment, the nucleic-acid dose is less than about 1 mg/cm² of tissue or less than about 1 mg/100,000 cells or less than about 10 mg/kg.

Reprogrammed cells produced according to certain embodiments of the present invention are suitable for therapeutic and/or cosmetic applications as they do not contain undesirable exogenous DNA sequences, and they are not exposed to animal-derived or human-derived products, which may be undefined, and which may contain toxic and/or pathogenic contaminants. Furthermore, the high speed, efficiency, and reliability of certain embodiments of the present invention may reduce the risk of acquisition and accumulation of mutations and other chromosomal abnormalities. Certain embodiments of the present invention can thus be used to generate cells that have a safety profile adequate for use in therapeutic and/or cosmetic applications. For example, reprogramming cells using RNA and the medium of the present invention, wherein the medium does not contain animal or human-derived components, can yield cells that have not been exposed to allogeneic material. Certain embodiments are therefore directed to a reprogrammed cell that has a desirable safety profile. In one embodiment, the reprogrammed cell has a normal karyotype. In another embodiment, the reprogrammed cell has fewer than about 5 copy-number variations (CNVs) relative to the patient genome, such as fewer than about 3 copy-number variations relative to the patient genome, or no copy-number variations relative to the patient genome. In yet another embodiment, the reprogrammed cell has a normal karyotype and fewer than about 100 single nucleotide variants in coding regions relative to the patient genome, or fewer than about 50 single nucleotide variants in coding regions relative to the patient genome, or fewer than about 10 single nucleotide variants in coding regions relative to the patient genome.

Endotoxins and nucleases can co-purify and/or become associated with other proteins, such as serum albumin. Recombinant proteins, in particular, can often have high levels of associated endotoxins and nucleases, due in part to the lysis of cells that can take place during their production. Endotoxins and nucleases can be reduced, removed, replaced or otherwise inactivated by many of the methods of the present invention, including, for example, by acetylation, by addition of a stabilizer such as sodium octanoate, followed by heat treatment, by the addition of nuclease inhibitors to the albumin solution and/or medium, by crystallization, by contacting with one or more ion-exchange resins, by contacting with charcoal, by preparative electrophoresis or by affinity chromatography. It has now been discovered that partially or completely reducing, removing, replacing or otherwise inactivating endotoxins and/or nucleases from a medium and/or from one or more components of a medium can increase the efficiency with which cells can be transfected and reprogrammed. Certain embodiments are therefore directed to a method for transfecting a cell in vivo with one or more nucleic acids, wherein the transfection medium is treated to partially or completely reduce, remove, replace or otherwise inactivate one or more endotoxins and/or nucleases. Other embodiments are directed to a medium that causes minimal degradation of nucleic acids. In one embodiment, the medium contains less than about 1 EU/mL, or less than about 0.1 EU/mL, or less than about 0.01 EU/mL.

In certain situations, protein-based lipid carriers such as serum albumin can be replaced with non-protein-based lipid carriers such as methyl-beta-cyclodextrin. The medium of the present invention can also be used without a lipid carrier, for example, when transfection is performed using a method that may not require or may not benefit from the presence of a lipid carrier, for example, using one or more lipid-based transfection reagents, polymer-based transfection reagents or peptide-based transfection reagents or using electroporation. Many protein-associated molecules, such as metals, can be highly toxic to cells in vivo. This toxicity can cause decreased viability, as well as the acquisition of mutations. Certain embodiments thus have the additional benefit of producing cells that are free from toxic molecules.

The associated-molecule component of a protein can be measured by suspending the protein in solution and measuring the conductivity of the solution. Certain embodiments are therefore directed to a medium that contains a protein, wherein about a 10% solution of the protein in water has a conductivity of less than about 500 μmho/cm. In one embodiment, the solution has a conductivity of less than about 50 μmho/cm. In another embodiment, less than about 0.65% of the dry weight of the protein comprises lipids and/or less than about 0.35% of the dry weight of the protein comprises free fatty acids.

The amount of nucleic acid delivered to cells in vivo can be increased to increase the desired effect of the nucleic acid. However, increasing the amount of nucleic acid delivered to cells in vivo beyond a certain point can cause a decrease in the viability of the cells, due in part to toxicity of the transfection reagent. It has now been discovered that when a nucleic acid is delivered to a population of cells in vivo in a fixed volume (for example, cells in a region of tissue), the amount of nucleic acid delivered to each cell can depend on the total amount of nucleic acid delivered to the population of cells and to the density of the cells, with a higher cell density resulting in less nucleic acid being delivered to each cell. In certain embodiments, a cell in vivo is transfected with one or more nucleic acids more than once. Under certain conditions, for example when the cells are proliferating, the cell density may change from one transfection to the next. Certain embodiments are therefore directed to a method for transfecting a cell in vivo with a nucleic acid, wherein the cell is transfected more than once, and wherein the amount of nucleic acid delivered to the cell is different for two of the transfections. In one embodiment, the cell proliferates between two of the transfections, and the amount of nucleic acid delivered to the cell is greater for the second of the two transfections than for the first of the two transfections. In another embodiment, the cell is transfected more than twice, and the amount of nucleic acid delivered to the cell is greater for the second of three transfections than for the first of the same three transfections, and the amount of nucleic acid delivered to the cells is greater for the third of the same three transfections than for the second of the same three transfections. In yet another embodiment, the cell is transfected more than once, and the maximum amount of nucleic acid delivered to the cell during each transfection is sufficiently low to yield at least about 80% viability for at least two consecutive transfections.

It has now been discovered that modulating the amount of nucleic acid delivered to a population of proliferating cells in vivo in a series of transfections can result in both an increased effect of the nucleic acid and increased viability of the cells. It has been further discovered that, in certain situations, when cells in vivo are contacted with one or more nucleic acids encoding one or more reprogramming factors in a series of transfections, the efficiency of reprogramming can be increased when the amount of nucleic acid delivered in later transfections is greater than the amount of nucleic acid delivered in earlier transfections, for at least part of the series of transfections. Certain embodiments are therefore directed to a method for reprogramming a cell in vivo, wherein one or more nucleic acids is repeatedly delivered to the cell in a series of transfections, and the amount of the nucleic acid delivered to the cell is greater for at least one later transfection than for at least one earlier transfection. In one embodiment, the cell is transfected between about 2 and about 10 times, or between about 3 and about 8 times, or between about 4 and about 6 times. In another embodiment, the one or more nucleic acids includes at least one RNA molecule, the cell is transfected between about 2 and about 10 times, and the amount of nucleic acid delivered to the cell in each transfection is the same as or greater than the amount of nucleic acid delivered to the cell in the most recent previous transfection. In yet another embodiment, the amount of nucleic acid delivered to the cell in the first transfection is between about 20 ng/cm² and about 250 ng/cm², or between 100 ng/cm² and 600 ng/cm². In yet another embodiment, the cell is transfected about 5 times at intervals of between about 12 and about 48 hours, and the amount of nucleic acid delivered to the cell is about 25 ng/cm² for the first transfection, about 50 ng/cm² for the second transfection, about 100 ng/cm² for the third transfection, about 200 ng/cm² for the fourth transfection, and about 400 ng/cm² for the fifth transfection. In yet another embodiment, the cell is further transfected at least once after the fifth transfection, and the amount of nucleic acid delivered to the cell is about 400 ng/cm².

Certain embodiments are directed to a method for transfecting a cell in vivo with a nucleic acid, wherein the amount of nucleic acid is determined by measuring the cell density, and choosing the amount of nucleic acid to transfect based on the measurement of cell density. In one embodiment, the cell density is measured by optical means. In another embodiment, the cell is transfected repeatedly, the cell density increases between two transfections, and the amount of nucleic acid transfected is greater for the second of the two transfections than for the first of the two transfections.

It has now been discovered that, in certain situations, the amount of a circulating protein that is produced in a patient can be increased by administering to a patient a nucleic acid at a plurality of administration sites. In certain embodiments, the amount of a circulating protein is increased relative to the amount of the circulating protein that is produced in a patient by administering to the patient the nucleic acid at a single injection site. In one embodiment, the administering is by injection. In another embodiment, the injection is intradermal injection. In still another embodiment, the injection is subcutaneous or intramuscular injection. In some embodiments, the plurality of administration sites comprise administration sites in the skin. In other embodiments, the plurality of administration sites are at least about 1 or at least about 2 or at least about 5 or at least about 10 or at least about 20 or at least about 50 or at least about 100 administration sites. In one embodiment, the administering is performed within at least about 5 minutes or at least about 10 minutes or at least about 30 minutes or at least about 1 hour or at least about 2 hours or at least about 5 hours or at least about 12 hours or at least about 1 day. In certain embodiments, the amount of a circulating protein is increased by at least about 10 percent or at least about 20 percent or at least about 50 percent or at least about 100 percent or at least about 3-fold or at least about 5-fold or at least about 10-fold or at least about 20-fold or at least about 50-fold or at least about 100-fold or at least about 500-fold or at least about 1000-fold or greater than 1000-fold.

It has now been discovered that, in certain situations, the in vivo transfection efficiency and viability of cells contacted with the medium of the present invention can be improved by conditioning the medium. Certain embodiments are therefore directed to a method for conditioning a medium. Other embodiments are directed to a medium that is conditioned. In one embodiment, the feeders are fibroblasts, and the medium is conditioned for approximately 24 hours. Other embodiments are directed to a method for transfecting a cell in vivo, wherein the transfection medium is conditioned. Other embodiments are directed to a method for reprogramming and/or gene-editing a cell in vivo, wherein the medium is conditioned. In one embodiment, the feeders are mitotically inactivated, for example, by exposure to a chemical such as mitomycin-C or by exposure to gamma radiation. In certain embodiments, it may be beneficial to use only autologous materials, in part to, for example and not wishing to be bound by theory, avoid the risk of disease transmission from the feeders to the cell or the patient. Certain embodiments are therefore directed to a method for transfecting a cell in vivo, wherein the transfection medium is conditioned, and wherein the feeders are derived from the same individual as the cell being transfected. Other embodiments are directed to a method for reprogramming and/or gene-editing a cell in vivo, wherein the medium is conditioned, and wherein the feeders are derived from the same individual as the cell being reprogrammed and/or gene-edited.

Several molecules can be added to media by conditioning. Certain embodiments are therefore directed to a medium that is supplemented with one or more molecules that are present in a conditioned medium. In one embodiment, the medium is supplemented with Wnt1, Wnt2, Wnt3, Wnt3a or a biologically active fragment, analogue, variant, agonist, or family-member thereof. In another embodiment, the medium is supplemented with TGF-β or a biologically active fragment, analogue, variant, agonist, or family-member thereof. In yet another embodiment, a cell in vivo is reprogrammed according to the method of the present invention, wherein the medium is not supplemented with TGF-β for between about 1 and about 5 days, and is then supplemented with TGF-β for at least about 2 days. In yet another embodiment, the medium is supplemented with IL-6, IL-6R or a biologically active fragment, analogue, variant, agonist, or family-member thereof. In yet another embodiment, the medium is supplemented with a sphingolipid or a fatty acid. In still another embodiment, the sphingolipid is lysophosphatidic acid, lysosphingomyelin, sphingosine-1-phosphate or a biologically active analogue, variant or derivative thereof.

In addition to mitotically inactivating cells, under certain conditions, irradiation can change the gene expression of cells, causing cells to produce less of certain proteins and more of certain other proteins that non-irradiated cells, for example, members of the Wnt family of proteins. In addition, certain members of the Wnt family of proteins can promote the growth and transformation of cells. It has now been discovered that, in certain situations, the efficiency of reprogramming can be greatly increased by contacting a cell in vivo with a medium that is conditioned using irradiated feeders instead of mitomycin-c-treated feeders. It has been further discovered that the increase in reprogramming efficiency observed when using irradiated feeders is caused in part by Wnt proteins that are secreted by the feeders. Certain embodiments are therefore directed to a method for reprogramming a cell in vivo, wherein the cell is contacted with Wnt1, Wnt2, Wnt3, Wnt3a or a biologically active fragment, analogue, variant, family-member or agonist thereof, including agonists of downstream targets of Wnt proteins, and/or agents that mimic one or more of the biological effects of Wnt proteins, for example, 2-amino-4-[3,4-(methylenedioxy)benzylamino]-6-(3-methoxyphenyl)pyrimidine.

Because of the low efficiency of many DNA-based reprogramming methods, these methods may be difficult or impossible to use with cells derived from patient samples, which may contain only a small number of cells. In contrast, the high efficiency of certain embodiments of the present invention can allow reliable reprogramming of a small number of cells, including single cells. Certain embodiments are directed to a method for reprogramming a small number of cells. Other embodiments are directed to a method for reprogramming a single cell. In one embodiment, the cell is contacted with one or more enzymes. In another embodiment, the enzyme is collagenase. In yet another embodiment, the collagenase is animal-component free. In one embodiment, the collagenase is present at a concentration of between about 0.1 mg/mL and about 10 mg/mL, or between about 0.5 mg/mL and about 5 mg/mL. In another embodiment, the cell is a blood cell. In yet another embodiment, the cell is contacted with a medium containing one or more proteins that is derived from the patient's blood. In still another embodiment, the cell is contacted with a medium comprising: DMEM/F12+2 mM L-alanyl-L-glutamine+between about 5% and about 25% patient-derived serum, or between about 10% and about 20% patient-derived serum, or about 20% patient-derived serum.

It has now been discovered that, in certain situations, transfecting cells in vivo with a mixture of RNA encoding Oct4, Sox2, Klf4, and c-Myc using the medium of the present invention can cause the rate of proliferation of the cells to increase. When the amount of RNA delivered to the cells is too low to ensure that all of the cells are transfected, only a fraction of the cells may show an increased proliferation rate. In certain situations, such as when generating a personalized therapeutic, increasing the proliferation rate of cells may be desirable, in part because doing so can reduce the time necessary to generate the therapeutic, and therefore can reduce the cost of the therapeutic. Certain embodiments are therefore directed to a method for transfecting a cell in vivo with a mixture of RNA encoding Oct4, Sox2, Klf4, and c-Myc. In one embodiment, the cell exhibits an increased proliferation rate. In another embodiment, the cell is reprogrammed.

Many diseases are associated with one or more mutations. Mutations can be corrected by contacting a cell with a nucleic acid that encodes a protein that, either alone or in combination with other molecules, corrects the mutation (an example of gene-editing). Examples of such proteins include: a nuclease, a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease, a meganuclease, a nickase, a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein a DNA-repair protein, a DNA-modification protein, a base-modification protein, a DNA methyltransferase, an protein that causes DNA demethylation, an enzyme for which DNA is a substrate or a natural or engineered variant, family-member, orthologue, fragment or fusion construct thereof. Certain embodiments are therefore directed to a method for transfecting a cell in vivo with a nucleic acid, wherein the nucleic acid encodes a protein that, either alone or in combination with other molecules, creates a single-strand or double-strand break in a DNA molecule. In a one embodiment, the protein is a zinc finger nuclease or a TALEN. In another embodiment, the nucleic acid is an RNA molecule. In yet another embodiment, the single-strand or double-strand break is within about 5,000,000 bases of the transcription start site of a gene selected from the group: SERPINA1, CCR5, CXCR4, GAD1, GAD2, CFTR, HBA1, HBA2, HBB, HBD, FANCA, XPA, XPB, XPC, ERCC2, POLH, HTT, DMD, SOD1, APOE, PRNP, BRCA1, and BRCA2 or an analogue, variant or family-member thereof. In one embodiment, the present invention relates to gene-editing of the MYC protein (e.g. correcting one or more mutations that may be linked to cancer), optionally with a TALEN. In yet another embodiment, the cell is transfected with a nucleic acid that acts as a repair template by either causing the insertion of a DNA sequence in the region of the single-strand or double-strand break or by causing the DNA sequence in the region of the single-strand or double-strand break to otherwise change. In yet another embodiment, the gene-editing protein contains a DNA modification domain. In yet another embodiment, the gene-editing protein corrects a mutation without creating a single-strand break. In yet another embodiment, the gene-editing protein corrects a mutation without creating a double-strand break. In yet another embodiment, the gene-editing protein corrects a mutation by causing the replacement of one base with another base. In one embodiment, adenine is replaced by cytosine. In another embodiment, adenine is replaced by guanine. In yet another embodiment, adenine is replaced by thymine. In yet another embodiment, cytosine is replaced by adenine. In yet another embodiment, cytosine is replaced by guanine. In yet another embodiment, cytosine is replaced by thymine. In yet another embodiment, guanine is replaced by adenine. In yet another embodiment, guanine is replaced by cytosine. In yet another embodiment, guanine is replaced by thymine. In yet another embodiment, thymine is replaced by adenine. In yet another embodiment, thymine is replaced by cytosine. In yet another embodiment, thymine is replaced by guanine. In one embodiment, the replacement of one base with another base is a one-step process. In another embodiment, the replacement of one base with another base is a multi-step process. In some embodiments, one base is replaced by more than one base, for example, by two bases. In other embodiments, more than one base is replaced by one base. In still other embodiments, more than one base is replaced by more than one base. In some embodiments, the gene-editing protein contains a deaminase domain. In one embodiment the deaminase domain comprises a cytidine deaminase domain. In another embodiment, the deaminase domain comprises an adenosine deaminase domain. In yet another embodiment, the deaminase domain comprises a guanosine deaminase domain. In yet another embodiment, the gene-editing protein comprises a sequence that is at least about 50% or at least about 60% or at least about 70% or at least about 80% or at least about 90% or at least about 95% or at least about 99% homologous to one or more of: SEQ ID NOs: 587, 588, 589, 590, 591, 592, and 593. In yet another embodiment, the gene-editing protein comprises a linker. In one embodiment, the linker is a flexible linker. In another embodiment, the linker positions the deaminase domain in proximity to a target base. In yet another embodiment, the gene-editing protein deaminates the target base. In yet another embodiment, the gene-editing protein comprises a glycosylase-inhibitor domain. In yet another embodiment, the gene-editing protein comprises glycosylase-inhibitor activity. In one embodiment, the glycosylase inhibitor is a uracil glycosylase inhibitor. In another embodiment, the glycosylase inhibitor is a N-methylpurine DNA glycosylase inhibitor. In yet another embodiment, the cell is reprogrammed, and subsequently, the cell is gene-edited. In yet another embodiment, the cell is gene-edited, and subsequently, the cell is reprogrammed. In yet another embodiment, the gene-editing and reprogramming are performed within about 7 days of each other. In yet another embodiment, the gene-editing and reprogramming occur simultaneously or on the same day. In yet another embodiment, the cell is a skin cell, the skin cell is gene-edited to disrupt the CCR5 gene, the skin cell is reprogrammed to a hematopoietic stem cell, thus producing a therapeutic for HIV/AIDS, and the therapeutic is used to treat a patient with HIV/AIDS. In yet another embodiment, the skin cell is derived from the same patient whom the therapeutic is used to treat.

Certain embodiments are directed to methods and compositions for the treatment of rare diseases. In some embodiments, the rare disease is one or more of: a rare metabolic disease, a rare cardiovascular disease, a rare dermatologic disease, a rare neurologic disease, a rare developmental disease, a rare genetic disease, a rare pulmonary disease, a rare liver disease, a rare kidney disease, a rare psychiatric disease, a rare reproductive disease, a rare musculoskeletal disease, a rare orthopedic disease, an inborn error of metabolism, a lysosomal storage disease, and a rare ophthalmologic disease. In one embodiment, the disease is alpha-1-antitrypsin deficiency. Some embodiments are directed to a treatment comprising a nucleic acid encoding a gene-editing protein that is capable of causing a deletion in a gene that is associated with one or more of: a gain-of-function mutation, a loss-of-function mutation, a recessive mutation, a dominant mutation or a dominant negative mutation. Other embodiments are directed to a treatment comprising a nucleic acid encoding a gene-editing protein that is capable of correcting one or more of: a gain-of-function mutation, a loss-of-function mutation, a recessive mutation, a dominant mutation or a dominant negative mutation. In one embodiment, the treatment ameliorates one or more of the symptoms in a subject. In another embodiment, the subject is a human subject. In yet another embodiment, the subject is a veterinary subject. Some embodiments are directed to a treatment for alpha-1-antitrypsin deficiency comprising administering to a subject a nucleic acid comprising a gene-editing protein that is capable of causing a deletion in or near the SERPINA1 gene. Other embodiments are directed to a treatment for alpha-1-antitrypsin deficiency comprising administering to a subject a nucleic acid encoding a gene-editing protein that is capable of correcting a mutation in or near the SERPINA1 gene. In one embodiment the mutation is the Z mutation. In one embodiment, the deletion or correction reduces the accumulation of polymerized alpha-1-antitrypsin protein in the subject's cells and/or increases the secretion of alpha-1-antitrypsin from the subject's cells. In another embodiment, the treated cells regenerate a diseased organ. In yet another embodiment, the diseased organ is the liver. In yet another embodiment, the diseased organ is the lung. In yet another embodiment, the treatment delays or eliminates the subject's need for a liver and/or lung transplant. Other embodiments are directed to a treatment for epidermolysis bullosa. In one embodiment the epidermolysis bullosa is dystrophic epidermolysis bullosa. In another embodiment, the epidermolysis bullosa is epidermolysis bullosa simplex. In yet another embodiment, the dystrophic epidermolysis bullosa is recessive dystrophic epidermolysis bullosa. In some embodiments, the treatment comprises administering to a subject a nucleic acid encoding a gene-editing protein that is capable of correcting a mutation in or near the COL7A1 gene. In one embodiment, the correction increases the amount of functional collagen VII produced by the subject's cells. In another embodiment, the treatment reduces the size, severity, and/or frequency of recurrence of skin lesions and/or blisters. Still other embodiments are directed to a treatment for primary hyperoxaluria. In one embodiment the primary hyperoxaluria is type I primary hyperoxaluria. In another embodiment, the treatment comprises administering to a subject a nucleic acid encoding a gene-editing protein that is capable of correcting a mutation in or near the AGXT gene. In yet another embodiment, the treatment delays or eliminates the subject's need for a kidney and/or liver transplant.

Genes that can be edited according to the methods of the present invention to produce therapeutics of the present invention include genes that can be edited to restore normal function, as well as genes that can be edited to reduce or eliminate function. Such genes include, but are not limited to alpha-1-antitrypsin (SERPINA1), mutations in which can cause alpha-1-antitrypsin deficiency, beta globin (HBB), mutations in which can cause sickle cell disease (SCD) and β-thalassemia, breast cancer 1, early onset (BRCA1) and breast cancer 2, early onset (BRCA2), mutations in which can increase susceptibility to breast cancer, C-C chemokine receptor type 5 (CCR5) and C-X-C chemokine receptor type 4 (CXCR4), mutations in which can confer resistance to HIV infection, cystic fibrosis transmembrane conductance regulator (CFTR), mutations in which can cause cystic fibrosis, dystrophin (DMD), mutations in which can cause muscular dystrophy, including Duchenne muscular dystrophy and Becker's muscular dystrophy, glutamate decarboxylase 1 and glutamate decarboxylase 2 (GAD1, GAD2), mutations in which can prevent autoimmune destruction of β-cells, hemoglobin alpha 1, hemoglobin alpha 2, and hemoglobin delta (HBA1, HBA2, and HBD), mutations in which can cause thalassemia, desmoplakin, keratin 5, keratin 14, plectin, integrin alpha-6, integrin beta-4, laminin subunit alpha-3, laminin subunit beta-3, laminin subunit gamma-2, collagen type VII alpha 1, collagen type XVII alpha 1, and matrix metalloproteinase-1 (DSP, KRT5, KRT14, PLEC1, ITGA6, ITGB4, LAMA3, LAMB3, LAMC2, COL7A1, COL17A1, and MMP1), mutations in which can cause epidermolysis bullosa, Huntington (HTT), mutations in which can cause Huntington's disease, superoxide dismutase 1 (SOD1), mutations in which can cause amyotrophic lateral sclerosis (ALS), XPA, XPB, XPC, XPD (ERCC6) and polymerase (DNA directed), eta (POLH), mutations in which can cause xeroderma pigmentosum, leucine-rich repeat kinase 2 (LRRK2), mutations in which can cause Parkinson's disease, and Fanconi anemia, complementation groups A, B, C, D1, D2, E, F, G, I, J, L, M, N, P (FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, FANCN, FANCP), and RAD51 homolog C (S. cerevisiae) (RAD51C), mutations in which can cause Fanconi anemia.

In a specific embodiment, the present invention relates to method of modulating Growth-Factor-Differentiation-Factor-15 (GDF15). In a specific embodiment, there is provided a method of treatment in which an RNA as described herein is administered to modulate GDF15 levels. In some embodiments, the RNA encodes GDF15. In a specific embodiment, the gene that is edited is Growth-Factor-Differentiation-Factor-15 (GDF15). In various embodiments, the present invention relates to targeting and/or expressing GDF15 to treat or prevent a disease or disorder such as a metabolic disease or disorder (e.g., diabetes (type I and II), insulin resistance, obesity, dyslipidemia, hypercholesterolemia, hyperglycemia, hyperinsulinemia, hypertension, hepatosteaotosis such as non-alcoholic steatohepatitis (NASH) and non-alcoholic fatty acid liver disease (NAFLD), cancer, a disease or disorder associated with impaired lipid metabolism, a disease or disorder associated with impaired renal function (e.g., chronic kidney diseases, nephropathy such as diabetic nephropathy, kidney failure), a disease or disorder associated with impaired hepatic function, a disease or disorder associated with impaired lung function, a vascular or cardiovascular disease or disorder (e.g., coronary artery disease, cardiomyopathy, hypertension, atrial fibrillation, preeclampsia, peripheral artery disease, atherosclerosis, heart failure, acute myocardial infarction, acute coronary syndrome, muscle wasting, hypertensive ventricular hypertrophy, hypertensive cardiomyopathy, ischemic heart disease, myocardial infarction, abdominal aortic aneurysm, a blood clot, deep vein thrombosis, venous stasis disease, phlebitis, varicose veins etc.), muscle wasting, inflammation, and a respiratory disease. In a specific embodiment, targeting of GDF15 improves lipid metabolism by mobilizing lipid deposits from the liver and/or other tissues into circulation for use in metabolism.

In certain disorders, the body may increase GDF15 levels in an attempt to counteract one or more negative aspects of the disease state. Certain embodiments are therefore directed to increasing GDF15 levels. In one embodiment, the serum level of GDF15 is increased. In another embodiment, the level of GDF15 in a tissue and/or organ is increased. In yet another embodiment, ALT levels are reduced. In yet another embodiment, AST levels are reduced. In still another embodiment, glucose levels are reduced. In still another embodiment, serum cholesterol levels are increased. In still another embodiment, serum triglyceride levels are increased. In yet another embodiment, food intake is reduced. In yet another embodiment, bodyweight is reduced. In a further embodiment, treatment improves adherence to a diet and/or exercise regimen.

In a specific embodiment, expressing GDF15 improves lipid metabolism by mobilizing lipid deposits from the liver and/or other tissues into circulation for use in metabolism. In a specific embodiment, expressing and/or targeting of GDF15 reduces liver inflammation. In a specific embodiment, expressing and/or targeting of GDF15 treats or prevents one or more of fatty liver, NAFLD, NASH, inflammation, hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. In a specific embodiment, expressing and/or targeting of GDF15 treats or prevents one or more of chronic kidney disease, acute kidney injury, nephropathy, diabetic nephropathy, kidney failure, and kidney fibrosis. In a specific embodiment, expressing and/or targeting GDF15 in combination with expressing and/or targeting another member of the TGF-β superfamily improves lipid metabolism by mobilizing lipid deposits from the liver and/or other tissues into circulation for use in metabolism, reduces liver inflammation, and/or treats or prevents one or more of fatty liver, NAFLD, NASH, inflammation, hepatitis, fibrosis, cirrhosis, hepatocellular carcinoma, chronic kidney disease, acute kidney injury, nephropathy, diabetic nephropathy, kidney failure, and kidney fibrosis. Certain embodiments are directed to a therapeutic comprising a nucleic acid. In one embodiment, the nucleic acid encodes one or more gene-editing proteins. Other embodiments are directed to a therapeutic comprising one or more cells that are transfected, reprogrammed, and/or gene-edited in vivo according to the methods of the present invention. In one embodiment, a cell is transfected, reprogrammed, and/or gene-edited, and the transfected, reprogrammed, and/or gene-edited cell is introduced into a patient. In another embodiment, the cell is harvested from the same patient into whom the transfected, reprogrammed and/or gene-edited cell is introduced. Examples of diseases that can be treated with therapeutics of the present invention include, but are not limited to Alzheimer's disease, spinal cord injury, amyotrophic lateral sclerosis, cystic fibrosis, heart disease, including ischemic and dilated cardiomyopathy, macular degeneration, Parkinson's disease, Huntington's disease, diabetes, sickle-cell anemia, thalassemia, Fanconi anemia, xeroderma pigmentosum, muscular dystrophy, severe combined immunodeficiency, hereditary sensory neuropathy, cancer, and HIV/AIDS. In certain embodiments, the therapeutic comprises a cosmetic. In one embodiment, a cell is harvested from a patient, the cell is reprogrammed and expanded to a large number of adipose cells to produce a cosmetic, and the cosmetic is introduced into the patient. In still another embodiment, the cosmetic is used for tissue reconstruction.

While detailed examples are provided herein for the production of specific types of cells and for the production of therapeutics comprising specific types of cells, it is recognized that the methods of the present invention can be used to produce many other types of cells, and to produce therapeutics comprising one or more of many other types of cells, for example, by reprogramming a cell according to the methods of the present invention, and culturing the cell under conditions that mimic one or more aspects of development by providing conditions that resemble the conditions present in the cellular microenvironment during development.

Certain embodiments are directed to a library of cells with a variety of human leukocyte antigen (HLA) types (“HLA-matched libraries”). An HLA-matched library may be beneficial in part because it can provide for the rapid production and/or distribution of therapeutics without the patient having to wait for a therapeutic to be produced from the patient's cells. Such a library may be particularly beneficial for the production of cosmetics and for the treatment of heart disease and diseases of the blood and/or immune system for which patients may benefit from the immediate availability of a therapeutic or cosmetic.

The DNA sequence of a cell can be altered by contacting the cell with a gene-editing protein or by inducing the cell to express a gene-editing protein. However, previously disclosed gene-editing proteins suffer from low binding efficiency and excessive off-target activity, which can introduce undesired mutations in the DNA of the cell, severely limiting their use in vivo, for example in therapeutic and cosmetic applications, in which the introduction of undesired mutations in a patient's cells could lead to the development of cancer. It has now been discovered that gene-editing proteins that comprise the StsI endonuclease cleavage domain (SEQ ID NO: 1) can exhibit substantially lower off-target activity in vivo than previously disclosed gene-editing proteins, while maintaining a high level of on-target activity in vivo. Other novel engineered proteins have also been discovered that can exhibit high on-target activity in vivo, low off-target activity in vivo, small size, solubility, and other desirable characteristics when they are used as the nuclease domain of a gene-editing protein: StsI-HA (SEQ ID NO: 2), StsI-HA2 (SEQ ID NO: 3), StsI-UHA (SEQ ID NO: 4), StsI-UHA2 (SEQ ID NO: 5), StsI-HF (SEQ ID NO: 6), and StsI-UHF (SEQ ID NO: 7). StsI-HA, StsI-HA2 (high activity), StsI-UHA, and StsI-UHA2 (ultra-high activity) can exhibit higher on-target activity in vivo than both wild-type StsI and wild-type FokI, due in part to specific amino-acid substitutions within the N-terminal region at the 34 and 61 positions, while StsI-HF (high fidelity) and StsI-UHF (ultra-high fidelity) can exhibit lower off-target activity in vivo than both wild-type StsI and wild-type FokI, due in part to specific amino-acid substitutions within the C-terminal region at the 141 and 152 positions.

Certain embodiments are therefore directed to a protein. In some embodiments, the protein is present in vivo. In other embodiments, the protein comprises a nuclease domain. In one embodiment, the nuclease domain comprises one or more of: the cleavage domain of FokI endonuclease (SEQ ID NO: 53), the cleavage domain of StsI endonuclease (SEQ ID NO: 1), StsI-HA (SEQ ID NO: 2), StsI-HA2 (SEQ ID NO: 3), StsI-UHA (SEQ ID NO: 4), StsI-UHA2 (SEQ ID NO: 5), StsI-HF (SEQ ID NO: 6), and StsI-UHF (SEQ ID NO: 7) or a biologically active fragment or variant thereof.

It has also been discovered that engineered gene-editing proteins that comprise DNA-binding domains comprising certain novel repeat sequences can exhibit lower off-target activity in vivo than previously disclosed gene-editing proteins, while maintaining a high level of on-target activity in vivo. Certain of these engineered gene-editing proteins can provide several advantages over previously disclosed gene-editing proteins, including, for example, increased flexibility of the linker region connecting repeat sequences, which can result in increased binding efficiency. Certain embodiments are therefore directed to a protein comprising a plurality of repeat sequences. In one embodiment, at least one of the repeat sequences contains the amino-acid sequence: GabG, where “a” and “b” each represent any amino acid. In one embodiment, the protein is a gene-editing protein. In another embodiment, one or more of the repeat sequences are present in a DNA-binding domain. In a further embodiment, “a” and “b” are each independently selected from the group: H and G. In a still further embodiment, “a” and “b” are H and G, respectively. In one embodiment, the amino-acid sequence is present within about 5 amino acids of the C-terminus of the repeat sequence. In another embodiment, the amino-acid sequence is present at the C-terminus of the repeat sequence. In some embodiments, one or more G in the amino-acid sequence GabG is replaced with one or more amino acids other than G, for example A, H or GG. In one embodiment, the repeat sequence has a length of between about 32 and about 40 amino acids or between about 33 and about 39 amino acids or between about 34 and 38 amino acids or between about 35 and about 37 amino acids or about 36 amino acids or greater than about 32 amino acids or greater than about 33 amino acids or greater than about 34 amino acids or greater than about 35 amino acids. Other embodiments are directed to a protein comprising one or more transcription activator-like effector domains. In one embodiment, at least one of the transcription activator-like effector domains comprises a repeat sequence. Other embodiments are directed to a protein comprising a plurality of repeat sequences generated by inserting one or more amino acids between at least two of the repeat sequences of a transcription activator-like effector domain. In one embodiment, one or more amino acids is inserted about 1 or about 2 or about 3 or about 4 or about 5 amino acids from the C-terminus of at least one repeat sequence. Still other embodiments are directed to a protein comprising a plurality of repeat sequences, wherein about every other repeat sequence has a different length than the repeat sequence immediately preceding or following the repeat sequence. In one embodiment, every other repeat sequence is about 36 amino acids long. In another embodiment, every other repeat sequence is 36 amino acids long. Still other embodiments are directed to a protein comprising a plurality of repeat sequences, wherein the plurality of repeat sequences comprises at least two repeat sequences that are each at least 36 amino acids long, and wherein at least two of the repeat sequences that are at least 36 amino acids long are separated by at least one repeat sequence that is less than 36 amino acids long. Some embodiments are directed to a protein that comprises one or more sequences selected from, for example, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, and SEQ ID NO: 60.

Other embodiments are directed to a protein that comprises a DNA-binding domain. In some embodiments, the DNA-binding domain comprises a plurality of repeat sequences. In one embodiment, the plurality of repeat sequences enables high-specificity recognition of a binding site in a target DNA molecule. In another embodiment, at least two of the repeat sequences have at least about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 98%, or about 99% homology to each other. In a further embodiment, at least one of the repeat sequences comprises one or more regions capable of binding to a binding site in a target DNA molecule. In a still further embodiment, the binding site comprises a defined sequence of between about 1 to about 5 bases in length. In one embodiment, the DNA-binding domain comprises a zinc finger. In another embodiment, the DNA-binding domain comprises a transcription activator-like effector (TALE). In a further embodiment, the plurality of repeat sequences includes at least one repeat sequence having at least about 50% or about 60% or about 70% or about 80% or about 90% or about 95% or about 98%, or about 99% homology to a TALE. In a still further embodiment, the gene-editing protein comprises a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein. In one embodiment, the gene-editing protein comprises a nuclear-localization sequence. In another embodiment, the nuclear-localization sequence comprises the amino-acid sequence: PKKKRKV (SEQ ID NO: 471). In one embodiment, the gene-editing protein comprises a mitochondrial-localization sequence. In another embodiment, the mitochondrial-localization sequence comprises the amino-acid sequence: LGRVIPRKIASRASLM (SEQ ID NO: 472). In one embodiment, the gene-editing protein comprises a linker. In another embodiment, the linker connects a DNA-binding domain to a nuclease domain. In a further embodiment, the linker is between about 1 and about 10 amino acids long. In some embodiments, the linker is about 1, about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10 amino acids long. In one embodiment, the gene-editing protein is capable of generating a nick or a double-strand break in a target DNA molecule.

Certain embodiments are directed to a method for modifying the genome of a cell in vivo, the method comprising introducing into a cell in vivo a nucleic acid molecule encoding a non-naturally occurring fusion protein comprising an artificial transcription activator-like (TAL) effector repeat domain comprising one or more repeat units 36 amino acids in length and an endonuclease domain, wherein the repeat domain is engineered for recognition of a predetermined nucleotide sequence, and wherein the fusion protein recognizes the predetermined nucleotide sequence. In one embodiment, the cell is a eukaryotic cell. In another embodiment, the cell is an animal cell. In a further embodiment, the cell is a mammalian cell. In a still further embodiment, the cell is a human cell. In one embodiment, the cell is a plant cell. In another embodiment, the cell is a prokaryotic cell. In some embodiments, the fusion protein introduces an endonucleolytic cleavage in a nucleic acid of the cell, whereby the genome of the cell is modified.

Certain embodiments are directed to a composition for altering the DNA sequence of a cell in vivo comprising a nucleic acid, wherein the nucleic acid encodes a gene-editing protein. Other embodiments are directed to a composition for altering the DNA sequence of a cell in vivo comprising a nucleic-acid mixture, wherein the nucleic-acid mixture comprises: a first nucleic acid that encodes a first gene-editing protein, and a second nucleic acid that encodes a second gene-editing protein. In one embodiment, the binding site of the first gene-editing protein and the binding site of the second gene-editing protein are present in the same target DNA molecule. In another embodiment, the binding site of the first gene-editing protein and the binding site of the second gene-editing protein are separated by less than about 50 bases, or less than about 40 bases, or less than about 30 bases or less than about 20 bases, or less than about 10 bases, or between about 10 bases and about 25 bases or about 15 bases. In one embodiment, the nuclease domain of the first gene-editing protein and the nuclease domain of the second gene-editing protein are capable of forming a dimer. In another embodiment, the dimer is capable of generating a nick or double-strand break in a target DNA molecule.

Certain embodiments are directed to a therapeutic composition. Other embodiments are directed to a cosmetic composition. In some embodiments, the composition comprises a repair template. In a further embodiment, the repair template is a single-stranded DNA molecule or a double-stranded DNA molecule.

Other embodiments are directed to an article of manufacture for synthesizing a protein or a nucleic acid encoding a protein. In one embodiment, the article is a nucleic acid. In another embodiment, the protein comprises a DNA-binding domain. In a further embodiment, the nucleic acid comprises a nucleotide sequence encoding a DNA-binding domain. In one embodiment, the protein comprises a nuclease domain. In another embodiment, the nucleic acid comprises a nucleotide sequence encoding a nuclease domain. In one embodiment, the protein comprises a plurality of repeat sequences. In another embodiment, the nucleic acid encodes a plurality of repeat sequences. In a further embodiment, the nuclease domain is selected from: FokI, StsI, StsI-HA, StsI-HA2, StsI-UHA, StsI-UHA2, StsI-HF, and StsI-UHF or a natural or engineered variant or biologically active fragment thereof. In one embodiment, the nucleic acid comprises an RNA-polymerase promoter. In another embodiment, the RNA-polymerase promoter is a T7 promoter or a SP6 promoter. In a further embodiment, the nucleic acid comprises a viral promoter. In one embodiment, the nucleic acid comprises an untranslated region. In another embodiment, the nucleic acid is an in vitro-transcription template.

Certain embodiments are directed to a method for inducing a cell to express a protein in vivo. Other embodiments are directed to a method for altering the DNA sequence of a cell in vivo comprising transfecting the cell in vivo with a gene-editing protein or inducing the cell to express a gene-editing protein in vivo. Still other embodiments are directed to a method for reducing the expression of a protein of interest in a cell in vivo. In one embodiment, the cell is induced to express a gene-editing protein, wherein the gene-editing protein is capable of creating a nick or a double-strand break in a target DNA molecule. In another embodiment, the nick or double-strand break results in inactivation of a gene. Still other embodiments are directed to a method for generating an inactive, reduced-activity or dominant-negative form of a protein in vivo. In one embodiment, the protein is survivin. Still other embodiments are directed to a method for repairing one or more mutations in a cell in vivo. In one embodiment, the cell is contacted with a repair template. In another embodiment, the repair template is a DNA molecule. In a further embodiment, the repair template does not contain a binding site of the gene-editing protein. In a still further embodiment, the repair template encodes an amino-acid sequence that is encoded by a DNA sequence that comprises a binding site of the gene-editing protein.

In various embodiments, the repair template is about 20 nucleotides, or about 30 nucleotides, or about 40 nucleotides, or about 50 nucleotides, or about 60 nucleotides, or about 70 nucleotides, or about 80 nucleotides, or about 90 nucleotides, or about 100 nucleotides, or about 150 nucleotides, or about 200 nucleotides, or about 300 nucleotides, or about 400 nucleotides, or about 500 nucleotides, or about 750 nucleotides, or about 1000 nucleotides. In various embodiments, the repair template is about 20-1000 nucleotides, or about 20-500 nucleotides, or about 20-400 nucleotides, or about 20-200 nucleotides, or about 20-100 nucleotides, or about 80-100 nucleotides, or about 50-100 nucleotides.

In various embodiments, the mass ratio of RNA (e.g. synthetic RNA encoding gene-editing protein) to repair template is about 1:10, or about 1:9, or about 1:8, or about 1:7, or about 1:6, or about 1:5, or about 1:4, or about 1:3, or about 1:2, or about 1:1, or about 2:1, or about 3:1, or about 4:1, or about 5:1, or about 6:1, or about 7:1, or about 8:1, or about 9:1, or about 10:1.

In various embodiments, the molar ratio of RNA (e.g. synthetic RNA encoding gene-editing protein) to repair template is about 1:10, or about 1:9, or about 1:8, or about 1:7, or about 1:6, or about 1:5, or about 1:4, or about 1:3, or about 1:2, or about 1:1, or about 2:1, or about 3:1, or about 4:1, or about 5:1, or about 6:1, or about 7:1, or about 8:1, or about 9:1, or about 10:1.

In various embodiments, the repair template has a dual function, causing a repair to a gene-edited target sequence and preventing further binding of a gene-editing protein, thereby reducing or eliminating further gene-editing (e.g. via the repair template causing a repair that renders what was the gene-editing protein binding site no longer suitable for gene-editing protein binding). Accordingly, in some embodiments, the present gene-editing methods are tunable to ensure a single gene-edit per target site.

Other embodiments are directed to a method for treating a patient comprising administering to the patient a therapeutically or cosmetically effective amount of a protein or a nucleic acid encoding a protein. In one embodiment, the treatment results in one or more of the patient's symptoms being ameliorated. Certain embodiments are directed to a method for treating a patient comprising: a. inducing a cell to express a protein of interest by transfecting the cell in vivo with a nucleic acid encoding the protein of interest and/or b. reprogramming the cell in vivo. In one embodiment, the cell is reprogrammed to a less differentiated state. In another embodiment, the cell is reprogrammed by transfecting the cell with one or more synthetic RNA molecules encoding one or more reprogramming proteins. In a further embodiment, the cell is differentiated. In a still further embodiment, the cell is differentiated into one of: a skin cell, a glucose-responsive insulin-producing cell, a hematopoietic cell, a cardiac cell, a retinal cell, a renal cell, a neural cell, a stromal cell, a fat cell, a bone cell, a muscle cell, an oocyte, and a sperm cell. Other embodiments are directed to a method for treating a patient comprising: a. inducing a cell to express a gene-editing protein by transfecting the cell in vivo with a nucleic acid encoding a gene-editing protein and/or b. reprogramming the cell in vivo.

Other embodiments are directed to a complexation medium. In one embodiment, the complexation medium has a pH greater than about 7, or greater than about 7.2, or greater than about 7.4, or greater than about 7.6, or greater than about 7.8, or greater than about 8.0, or greater than about 8.2, or greater than about 8.4, or greater than about 8.6, or greater than about 8.8, or greater than about 9.0. In another embodiment, the complexation medium comprises transferrin. In a further embodiment, the complexation medium comprises DMEM. In a still further embodiment, the complexation medium comprises DMEM/F12. Still other embodiments are directed to a method for forming nucleic-acid-transfection-reagent complexes. In one embodiment, the transfection reagent is incubated with a complexation medium. In another embodiment, the incubation occurs before a mixing step. In a further embodiment, the incubation step is between about 5 seconds and about 5 minutes or between about 10 seconds and about 2 minutes or between about 15 seconds and about 1 minute or between about 30 seconds and about 45 seconds. In one embodiment, the transfection reagent is selected from Table 1. In another embodiment, the transfection reagent is a lipid or lipidoid. In a further embodiment, the transfection reagent comprises a cation. In a still further embodiment, the cation is a multivalent cation. In a still further embodiment, the transfection reagent is N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (a.k.a. MVL5) or a derivative thereof.

Certain embodiments are directed to a method for inducing a cell to express a protein by contacting the cell with a nucleic acid in vivo. In one embodiment, the cell is a mammalian cell. In another embodiment, the cell is a human cell or a rodent cell. Other embodiments are directed to a cell produced using one or more of the methods of the present invention. In one embodiment, the cell is present in a patient. In another embodiment, the cell is isolated from a patient. Other embodiments are directed to a screening library comprising a cell produced using one or more of the methods of the present invention. In one embodiment, the screening library is used for at least one of: toxicity screening, including: cardiotoxicity screening, neurotoxicity screening, and hepatotoxicity screening, efficacy screening, high-throughput screening, high-content screening, and other screening.

Other embodiments are directed to a kit containing a nucleic acid. In one embodiment, the kit contains a delivery reagent (a.k.a. “transfection reagent”). In another embodiment, the kit is a reprogramming kit. In a further embodiment, the kit is a gene-editing kit. Other embodiments are directed to a kit for producing nucleic acids. In one embodiment, the kit contains at least two of: pseudouridine-triphosphate, 5-methyluridine triphosphate, 5-methylcytidine triphosphate, 5-hydroxymethylcytidine triphosphate, N4-methylcytidine triphosphate, N4-acetylcytidine triphosphate, and 7-deazaguanosine triphosphate or one or more derivatives thereof. Other embodiments are directed to a therapeutic or cosmetic comprising a nucleic acid. In one embodiment, the therapeutic or cosmetic is a pharmaceutical composition. In another embodiment, the pharmaceutical composition is formulated. In a further embodiment, the formulation comprises an aqueous suspension of liposomes. Example liposome components are set forth in Table 1, and are given by way of example, and not by way of limitation. In one embodiment, the liposomes include one or more polyethylene glycol (PEG) chains. In another embodiment, the PEG is PEG2000. In a further embodiment, the liposomes include 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) or a derivative thereof. In one embodiment, the therapeutic comprises one or more ligands. In another embodiment, the therapeutic comprises at least one of: androgen, CD30 (TNFRSF8), a cell-penetrating peptide, CXCR, estrogen, epidermal growth factor, EGFR, HER2, folate, insulin, insulin-like growth factor-I, interleukin-13, integrin, progesterone, stromal-derived-factor-1, thrombin, vitamin D, and transferrin or a biologically active fragment or variant thereof. Still other embodiments are directed to a therapeutic or cosmetic comprising a cell generated using one or more of the methods of the present invention. In one embodiment, the therapeutic is administered to a patient for the treatment of any of the diseases or disorders described herein, including by way of non-limitation, type 1 diabetes, heart disease, including ischemic and dilated cardiomyopathy, macular degeneration, Parkinson's disease, cystic fibrosis, sickle-cell anemia, thalassemia, Fanconi anemia, severe combined immunodeficiency, hereditary sensory neuropathy, xeroderma pigmentosum, Huntington's disease, muscular dystrophy, amyotrophic lateral sclerosis, Alzheimer's disease, cancer, and infectious diseases including: hepatitis and HIV/AIDS.

Other embodiments are directed to a method for reprogramming a cell in vivo. In one embodiment, the cell is reprogrammed by contacting the cell with one or more nucleic acids. In one embodiment, the cell is contacted with a plurality of nucleic acids encoding at least one of: Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, Lin28 protein or a biologically active fragment, variant or derivative thereof. In another embodiment, the cell is contacted with a plurality of nucleic acids encoding a plurality of proteins including: Oct4 protein, Sox2 protein, Klf4 protein, and c-Myc protein or one or more biologically active fragments, variants or derivatives thereof. Still other embodiments are directed to a method for gene editing a cell in vivo. In one embodiment, the cell is gene-edited by contacting the cell with one or more nucleic acids.

Certain embodiments are directed to a method for inducing a cell in vivo to express a protein of interest comprising contacting a cell in vivo with a solution comprising albumin that is treated with an ion-exchange resin or charcoal and one or more nucleic acid molecules, wherein at least one of the one or more nucleic acid molecules encodes a protein of interest. In one embodiment, the method results in the cell expressing the protein of interest. In another embodiment, the one or more nucleic acid molecules comprise a synthetic RNA molecule. In one embodiment, the cell is a skin cell. In another embodiment, the cell is a muscle cell. In yet another embodiment, the cell is a dermal fibroblast. In yet another embodiment, the cell is a myoblast. In one embodiment, the protein of interest is an extracellular matrix protein. In another embodiment, the protein of interest is selected from: elastin, collagen, laminin, fibronectin, vitronectin, lysyl oxidase, elastin binding protein, a growth factor, fibroblast growth factor, transforming growth factor beta, granulocyte colony-stimulating factor, a matrix metalloproteinase, an actin, fibrillin, microfibril-associated glycoprotein, a lysyl-oxidase-like protein, and platelet-derived growth factor. In one embodiment, the solution is delivered to the dermis. In another embodiment, the delivering is by injection. In yet another embodiment, the delivering is by injection using a microneedle array. In one embodiment, the solution further comprises a growth factor. In another embodiment, the growth factor is selected from: fibroblast growth factor and transforming growth factor beta. In yet another embodiment, the solution further comprises cholesterol. Other embodiments are directed a method for inducing a cell in vivo to express a protein of interest comprising contacting a cell in vivo with a solution comprising cholesterol and one or more nucleic acid molecules, wherein at least one of the one or more nucleic acid molecules encodes a protein of interest. In one embodiment, the method results in the cell expressing the protein of interest. Still other embodiments are directed to a method for transfecting a cell in vivo with a nucleic acid molecule comprising contacting a cell in vivo with a solution comprising albumin that is treated with an ion-exchange resin or charcoal and a nucleic acid molecule. In one embodiment, the method results in the cell being transfected with the nucleic acid molecule. In another embodiment, the nucleic acid molecule is one of: a dsDNA molecule, a ssDNA molecule, a dsRNA molecule, a ssRNA molecule, a plasmid, an oligonucleotide, a synthetic RNA molecule, a miRNA molecule, an mRNA molecule, an siRNA molecule. Still other embodiments are directed to a method for treating a patient comprising delivering to a patient a composition comprising albumin that is treated with an ion-exchange resin or charcoal and one or more nucleic acid molecules, wherein at least one of the one or more nucleic acid molecules encodes a protein of interest. In one embodiment, the method results in the expression of the protein of interest in the patient. In another embodiment, the method results in the expression of the protein of interest in the dermis of the patient.

Certain embodiments are directed to a cosmetic composition comprising albumin that is treated with an ion-exchange resin or charcoal and a nucleic acid molecule. Other embodiments are directed to a cosmetic treatment article. In one embodiment, the cosmetic treatment article comprises a device configured to deliver a composition to a patient. In another embodiment, the nucleic acid molecule encodes elastin protein or collagen protein. Still other embodiments are directed to a solution for transfecting a cell in vivo comprising cholesterol or a cholesterol analog and one or more nucleic acid molecules. In one embodiment, the cholesterol or cholesterol analog is covalently bound to at least one of the one or more nucleic acid molecules. In another embodiment, the cholesterol analog is an oxysterol. In yet another embodiment, the cholesterol analog includes one or more of: an A-ring substitution, a B-ring substitution, a D-ring substitution, a side-chain substitution, a cholestanoic acid, a cholestenoic acid, a polyunsaturated moiety, a deuterated moiety, a fluorinated moiety, a sulfonated moiety, a phosphorylated moiety, and a fluorescent moiety. In yet another embodiment, the method comprises treating the patient with one or more of: a dermal filler, a neurotoxin (by way of illustration sodium channel inhibitors (e.g., tetrodotoxin), potassium channel inhibitors (e.g., tetraethylammonium), chloride channel inhibitors (e.g., chlorotoxin and curare), calcium channel inhibitors (e.g., conotoxin), synaptic vesicle release inhibitors (e.g., botulinum toxin and tetanus toxin) and blood brain barrier inhibitor (e.g., aluminum and mercury)) and a repair-inducing treatment.

Despite the tendency of transfection reagent nucleic acid complexes to precipitate, form clumps or otherwise degrade when stored for more than a few minutes, the present inventors have surprisingly discovered that transfection reagent nucleic acid complexes produced according to some embodiments of the present invention can be frozen and/or can be stored at various temperatures, including room temperature, about 4° C., about −20° C., about −80° C., and about −196° C. for an extended period of time, for example, for several hours, about 1 day, about 1 week, about 1 month, about 1 year, and longer than about 1 year. Some embodiments are therefore directed to a pharmaceutical formulation comprising synthetic RNA and a transfection reagent, wherein the pharmaceutical formulation is provided in solid form. Other embodiments are directed to a pharmaceutical formulation comprising synthetic RNA transfection reagent complexes, wherein the synthetic RNA transfection reagent complexes are provided in solid form. In various embodiments, the synthetic RNA transfection reagent complexes are provided in frozen form. Various embodiments are directed to a method for stabilizing nucleic acid transfection reagent complexes comprising forming nucleic acid transfection reagent complexes and contacting the nucleic acid transfection reagent complexes or vessel in which such are contained with a cryogenic liquid to produce stabilized nucleic acid transfection reagent complexes. In one embodiment, the nucleic acid transfection reagent complexes are stabilized for shipment or storage.

Illustrative subjects or patients refers to any vertebrate including, without limitation, humans and other primates (e.g., chimpanzees and other apes and monkey species), farm animals (e.g., cattle, sheep, pigs, goats, and horses), domestic mammals (e.g., dogs and cats), laboratory animals (e.g., rodents such as mice, rats, and guinea pigs), and birds (e.g., domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like). In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

Definitions

By “molecule” is meant a molecular entity (molecule, ion, complex, etc.).

By “RNA molecule” is meant a molecule that comprises RNA.

By “synthetic RNA molecule” is meant an RNA molecule that is produced outside of a cell or that is produced inside of a cell using bioengineering, by way of non-limiting example, an RNA molecule that is produced in an in vitro-transcription reaction, an RNA molecule that is produced by direct chemical synthesis or an RNA molecule that is produced in a genetically-engineered E. coli cell.

By “transfection” is meant contacting a cell with a molecule, wherein the molecule is internalized by the cell.

By “upon transfection” is meant during or after transfection.

By “transfection reagent” is meant a substance or mixture of substances that associates with a molecule and facilitates the delivery of the molecule to and/or internalization of the molecule by a cell, by way of non-limiting example, a cationic lipid, a charged polymer or a cell-penetrating peptide.

By “reagent-based transfection” is meant transfection using a transfection reagent.

By “medium” is meant a solvent or a solution comprising a solvent and a solute, by way of non-limiting example, Dulbecco's Modified Eagle's Medium (DMEM), DMEM+10% fetal bovine serum (FBS), saline or water.

By “complexation medium” is meant a medium to which a transfection reagent and a molecule to be transfected are added and in which the transfection reagent associates with the molecule to be transfected.

By “transfection medium” is meant a medium that can be used for transfection, by way of non-limiting example, Dulbecco's Modified Eagle's Medium (DMEM), DMEM/F12, saline or water.

By “recombinant protein” is meant a protein or peptide that is not produced in animals or humans. Non-limiting examples include human transferrin that is produced in bacteria, human fibronectin that is produced in an in vitro culture of mouse cells, and human serum albumin that is produced in a rice plant.

By “Oct4 protein” is meant a protein that is encoded by the POU5F1 gene, or a natural or engineered variant, family-member, orthologue, fragment or fusion construct thereof, by way of non-limiting example, human Oct4 protein (SEQ ID NO: 8), mouse Oct4 protein, Oct1 protein, a protein encoded by POU5F1 pseudogene 2, a DNA-binding domain of Oct4 protein or an Oct4-GFP fusion protein. In some embodiments the Oct4 protein comprises an amino acid sequence that has at least 70% identity with SEQ ID NO: 8, or in other embodiments, at least 75%, 80%, 85%, 90%, or 95% identity with SEQ ID NO: 8. In some embodiments, the Oct4 protein comprises an amino acid sequence having from 1 to 20 amino acid insertions, deletions, or substitutions (collectively) with respect to SEQ ID NO: 8. Or in other embodiments, the Oct4 protein comprises an amino acid sequence having from 1 to 15 or from 1 to 10 amino acid insertions, deletions, or substitutions (collectively) with respect to SEQ ID NO: 8.

By “Sox2 protein” is meant a protein that is encoded by the SOX2 gene, or a natural or engineered variant, family-member, orthologue, fragment or fusion construct thereof, by way of non-limiting example, human Sox2 protein (SEQ ID NO: 9), mouse Sox2 protein, a DNA-binding domain of Sox2 protein or a Sox2-GFP fusion protein. In some embodiments the Sox2 protein comprises an amino acid sequence that has at least 70% identity with SEQ ID NO: 9, or in other embodiments, at least 75%, 80%, 85%, 90%, or 95% identity with SEQ ID NO: 9.

In some embodiments, the Sox2 protein comprises an amino acid sequence having from 1 to 20 amino acid insertions, deletions, or substitutions (collectively) with respect to SEQ ID NO: 9. Or in other embodiments, the Sox2 protein comprises an amino acid sequence having from 1 to 15 or from 1 to 10 amino acid insertions, deletions, or substitutions (collectively) with respect to SEQ ID NO: 9.

By “Klf4 protein” is meant a protein that is encoded by the KLF4 gene, or a natural or engineered variant, family-member, orthologue, fragment or fusion construct thereof, by way of non-limiting example, human Klf4 protein (SEQ ID NO: 10), mouse Klf4 protein, a DNA-binding domain of Klf4 protein or a Klf4-GFP fusion protein. In some embodiments the Klf4 protein comprises an amino acid sequence that has at least 70% identity with SEQ ID NO: 10, or in other embodiments, at least 75%, 80%, 85%, 90%, or 95% identity with SEQ ID NO: 10. In some embodiments, the Klf4 protein comprises an amino acid sequence having from 1 to 20 amino acid insertions, deletions, or substitutions (collectively) with respect to SEQ ID NO: 10. Or in other embodiments, the Klf4 protein comprises an amino acid sequence having from 1 to 15 or from 1 to 10 amino acid insertions, deletions, or substitutions (collectively) with respect to SEQ ID NO: 10.

By “c-Myc protein” is meant a protein that is encoded by the MYC gene, or a natural or engineered variant, family-member, orthologue, fragment or fusion construct thereof, by way of non-limiting example, human c-Myc protein (SEQ ID NO: 11), mouse c-Myc protein, I-Myc protein, c-Myc (T58A) protein, a DNA-binding domain of c-Myc protein or a c-Myc-GFP fusion protein. In some embodiments the c-Myc protein comprises an amino acid sequence that has at least 70% identity with SEQ ID NO: 11, or in other embodiments, at least 75%, 80%, 85%, 90%, or 95% identity with SEQ ID NO: 11. In some embodiments, the c-Myc protein comprises an amino acid having from 1 to 20 amino acid insertions, deletions, or substitutions (collectively) with respect to SEQ ID NO: 11. Or in other embodiments, the c-Myc protein comprises an amino acid sequence having from 1 to 15 or from 1 to 10 amino acid insertions, deletions, or substitutions (collectively) with respect to SEQ ID NO: 11.

By “erythropoietin” or “erythropoietin protein” is meant a protein that is encoded by the EPO gene, or a natural or engineered variant, family-member, orthologue, fragment or fusion construct thereof, by way of non-limiting example, human erythropoietin (SEQ ID NO: 164), mouse erythropoietin, darbepoetin, darbepoetin alfa, NOVEPOETIN, a binding domain of erythropoietin or an erythropoietin-GFP fusion protein. In some embodiments the erythropoietin comprises an amino acid sequence that has at least 70% identity with SEQ ID NO: 164, or in other embodiments, at least 75%, 80%, 85%, 90%, or 95% identity with SEQ ID NO: 164. In some embodiments, the erythropoietin comprises an amino acid sequence having from 1 to 20 amino acid insertions, deletions, or substitutions (collectively) with respect to SEQ ID NO: 164. Or in other embodiments, the erythropoietin comprises an amino acid sequence having from 1 to 15 or from 1 to 10 amino acid insertions, deletions, or substitutions (collectively) with respect to SEQ ID NO: 164.

By “reprogramming” is meant causing a change in the phenotype of a cell, by way of non-limiting example, causing a β-cell progenitor to differentiate into a mature β-cell, causing a fibroblast to dedifferentiate into a pluripotent stem cell, causing a keratinocyte to transdifferentiate into a cardiac stem cell, causing the telomeres of a cell to lengthen or causing the axon of a neuron to grow.

By “reprogramming factor” is meant a molecule that, when a cell is contacted with the molecule and/or the cell expresses the molecule, can, either alone or in combination with other molecules, cause reprogramming, by way of non-limiting example, Oct4 protein, Tert protein or erythropoietin.

By “germ cell” is meant a sperm cell or an egg cell.

By “pluripotent stem cell” is meant a cell that can differentiate into cells of all three germ layers (endoderm, mesoderm, and ectoderm) in vivo.

By “somatic cell” is meant a cell that is not a pluripotent stem cell or a germ cell, by way of non-limiting example, a skin cell.

By “hematopoietic cell” is meant a blood cell or a cell that can differentiate into a blood cell, by way of non-limiting example, a hematopoietic stem cell or a white blood cell.

By “cardiac cell” is meant a heart cell or a cell that can differentiate into a heart cell, by way of non-limiting example, a cardiac stem cell or a cardiomyocyte.

By “retinal cell” is meant a cell of the retina or a cell that can differentiate into a cell of the retina, by way of non-limiting example, a retinal pigmented epithelial cell.

By “skin cell” is meant a cell that is normally found in the skin, by way of non-limiting example, a fibroblast, a keratinocyte, a melanocyte, an adipocyte, a mesenchymal stem cell, an adipose stem cell or a blood cell.

By “immunosuppressant” is meant a substance that can suppress one or more aspects of an immune system, and that is not normally present in a mammal, by way of non-limiting example, B18R or dexamethasone.

By “single-strand break” is meant a region of single-stranded or double-stranded DNA in which one or more of the covalent bonds linking the nucleotides has been broken in one of the one or two strands.

By “double-strand break” is meant a region of double-stranded DNA in which one or more of the covalent bonds linking the nucleotides has been broken in each of the two strands.

By “nucleotide” is meant a nucleotide or a fragment or derivative thereof, by way of non-limiting example, a nucleobase, a nucleoside, a nucleotide-triphosphate, etc.

By “nucleoside” is meant a nucleotide or a fragment or derivative thereof, by way of non-limiting example, a nucleobase, a nucleoside, a nucleotide-triphosphate, etc.

By “gene editing” is meant altering the DNA sequence of a cell, by way of non-limiting example, by transfecting the cell with a protein that causes a mutation in the DNA of the cell or by transfecting the cell with a protein that causes a chemical change in the DNA of the cell.

By “gene-editing protein” is meant a protein that can, either alone or in combination with one or more other molecules, alter the DNA sequence of a cell, by way of non-limiting example, a nuclease, a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease, a meganuclease, a nickase, a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein, a DNA-repair protein, a DNA-modification protein, a base-modification protein, a DNA methyltransferase, an protein that causes DNA demethylation, an enzyme for which DNA is a substrate or a natural or engineered variant, family-member, orthologue, domain, fragment or fusion construct thereof.

By “repair template” is meant a nucleic acid containing a region of at least about 70% homology with a sequence that is within 10 kb of a target site of a gene-editing protein.

By “repeat sequence” is meant an amino-acid sequence that is present in more than one copy in a protein, to within at least about 10% homology, by way of non-limiting example, a monomer repeat of a transcription activator-like effector.

By “DNA-binding domain” is meant a region of a molecule that is capable of binding to a DNA molecule, by way of non-limiting example, a protein domain comprising one or more zinc fingers, a protein domain comprising one or more transcription activator-like (TAL) effector repeat sequences or a binding pocket of a small molecule that is capable of binding to a DNA molecule.

By “binding site” is meant a nucleic-acid sequence that is capable of being recognized by a gene-editing protein, DNA-binding protein, DNA-binding domain or a biologically active fragment or variant thereof or a nucleic-acid sequence for which a gene-editing protein, DNA-binding protein, DNA-binding domain or a biologically active fragment or variant thereof has high affinity, by way of non-limiting example, an about 20-base-pair sequence of DNA in exon 1 of the human BIRC5 gene.

By “target” is meant a nucleic acid that contains a binding site.

Other definitions are set forth in U.S. application Ser. No. 13/465,490, U.S. Provisional Application No. 61/664,494, U.S. Provisional Application No. 61/721,302, International Application No. PCT/US12/67966, U.S. Provisional Application No. 61/785,404, U.S. Provisional Application No. 61/842,874, International Application No. PCT/US13/68118, U.S. Provisional Application No. 61/934,397, U.S. application Ser. No. 14/296,220, U.S. Provisional Application No. 62/038,608, U.S. Provisional Application No. 62/069,667, and International Application No. PCT/US2015/013949, the contents of which are hereby incorporated by reference in their entireties.

Selected Sequences SEQ ID NO Description 1 StsI 2 StsI-HA 3 StsI-HA2 4 StsI-UHA 5 StsI-UHA2 6 StsI-HF 7 StsI-UHF 8 Oct4 9 Sox2 10 Klf4 11 c-Myc 12 BIRC5_exon1 13 BIRC5_exon2 14 BIRC5_exon3 15 BIRC5_exon4 16 BIRC5-1.1-L 17 BIRC5-1.1-R 18 BIRC5-1.2-L 19 BIRC5-1.2-R 20 BIRC5-1.3-L 21 BIRC5-1.3-R 22 BIRC5-2.1-L 23 BIRC5-2.1-R 24 BIRC5-2.2-L 25 BIRC5-2.2-R 26 BIRC5-3.1-L 27 BIRC5-3.1-R 28 CDK1 29 CDK2 30 CDK3 31 CDK4 32 CDK5 33 CDK6 34 BIRC5 35 HIF1A 36 RRM2 37 KRAS 38 EGFR 39 MYC 40 PKN3 41 KIF11 42 APC 43 BRCA1 44 BRCA2 45 TP53 46 APP 47 HTT 48 IAPP 49 MAPT 50 PRNP 51 SNCA 52 SOD1 53 FokI 54 Repeat1 55 Repeat2 56 Repeat3 57 EO-GHGG-FokI (“GHGG” disclosed as SEQ ID NO: 547) 58 GHGG-FokI (“GHGG” disclosed as SEQ ID NO: 547) 59 EO-GHGG-StsI (“GHGG” disclosed as SEQ ID NO: 547) 60 GHGG-StsI (“GHGG” disclosed as SEQ ID NO: 547) 61 collagen alpha-1(I) chain preproprotein 62 collagen alpha-2(I) chain precursor 63 collagen alpha-1(II) chain isoform 1 precursor 64 collagen alpha-1(II) chain isoform 2 precursor 65 collagen alpha-1(III) chain preproprotein 66 collagen alpha-1(IV) chain preproprotein 67 collagen alpha-2(IV) chain preproprotein 68 collagen alpha-3(IV) chain precursor 69 collagen alpha-4(IV) chain precursor 70 collagen alpha-5(IV) chain isoform 1 precursor 71 collagen alpha-6(IV) chain isoform A precursor 72 collagen alpha-1(V) chain isoform 1 preproprotein 73 collagen alpha-2(V) chain preproprotein 74 collagen alpha-3(V) chain preproprotein 75 collagen alpha-1(VI) chain precursor 76 collagen alpha-2(VI) chain isoform 2C2 precursor 77 collagen alpha-3(VI) chain isoform 1 precursor 78 collagen alpha-1(VII) chain precursor 79 elastin isoform a precursor 80 elastin isoform b precursor 81 elastin isoform c precursor 82 elastin isoform d precursor 83 elastin isoform e precursor 84 elastin isoform f precursor 85 elastin isoform g precursor 86 elastin isoform h precursor 87 elastin isoform i precursor 88 elastin isoform j precursor 89 elastin isoform k precursor 90 elastin isoform l precursor 91 elastin isoform m precursor 92 protein-lysine 6-oxidase isoform 1 preproprotein 93 protein-lysine 6-oxidase isoform 2 94 telomerase reverse transcriptase isoform 1 95 telomerase reverse transcriptase isoform 2 96 fibronectin isoform 1 preproprotein 97 fibronectin isoform 3 preproprotein 98 fibronectin isoform 4 preproprotein 99 fibronectin isoform 5 preproprotein 100 fibronectin isoform 6 preproprotein 101 fibronectin isoform 7 preproprotein 102 vitronectin precursor 103 nidogen-1 precursor 104 laminin subunit alpha-1 precursor 105 insulin-like growth factor I isoform 1 preproprotein 106 fibroblast growth factor 1 isoform 1 precursor 107 fibroblast growth factor 2 108 transforming growth factor beta-1 precursor 109 transforming growth factor beta-2 isoform 1 precursor 110 transforming growth factor beta-2 isoform 2 precursor 111 actin, alpha skeletal muscle 112 actin, aortic smooth muscle 113 actin, cytoplasmic 1 114 actin, alpha cardiac muscle 1 proprotein 115 actin, cytoplasmic 2 116 actin, gamma-enteric smooth muscle isoform 1 precursor 117 actin, gamma-enteric smooth muscle isoform 2 precursor 118 granulocyte colony-stimulating factor isoform a precursor 119 granulocyte colony-stimulating factor isoform b precursor 120 granulocyte colony-stimulating factor isoform c precursor 121 granulocyte colony-stimulating factor isoform d precursor 122 platelet-derived growth factor subunit A isoform 1 preproprotein 123 platelet-derived growth factor subunit A isoform 2 preproprotein 124 platelet-derived growth factor subunit B isoform 1 preproprotein 125 platelet-derived growth factor subunit B isoform 2 preproprotein 126 platelet-derived growth factor C precursor 127 platelet-derived growth factor D isoform 1 precursor 128 platelet-derived growth factor D isoform 2 precursor 129 interstitial collagenase isoform 1 preproprotein 130 interstitial collagenase isoform 2 131 neutrophil collagenase preproprotein 132 stromelysin-2 preproprotein 133 macrophage metalloelastase preproprotein 134 fibrillin-1 precursor 135 fibrillin-2 precursor 136 lysyl oxidase homolog 1 preproprotein 137 lysyl oxidase homolog 2 precursor 138 lysyl oxidase homolog 3 isoform 1 precursor 139 lysyl oxidase homolog 3 isoform 2 precursor 140 lysyl oxidase homolog 3 isoform 3 141 lysyl oxidase homolog 4 precursor 142 microfibrillar-associated protein 2 isoform a precursor 143 microfibrillar-associated protein 2 isoform b precursor 144 microfibrillar-associated protein 5 precursor 145 disintegrin and metalloproteinase domain-containing protein 17 preprotein 146 desmoglein-2 preproprotein 147 DNA polymerase eta isoform 1 148 DNA polymerase eta isoform 2 149 DNA polymerase eta isoform 3 150 ferrochelatase, mitochondrial isoform a precursor 151 ferrochelatase, mitochondrial isoform b precursor 152 filaggrin 153 hyaluronan synthase 1 isoform 1 154 hyaluronan synthase 1 isoform 2 155 hyaluronan synthase 2 156 hyaluronan synthase 3 isoform a 157 hyaluronan synthase 3 isoform b 158 proopiomelanocortin 159 plakophilin-1 isoform 1a 160 plakophilin-1 isoform 1b 161 retinol dehydrogenase 10 162 mitochondrial brown fat uncoupling protein 1 163 tyrosinase precursor 164 erythropoietin 165 epoetin alfa 166 darbepoetin alfa 167 NOVEPOETIN 168 NOVECRIT

This invention is further illustrated by the following non-limiting examples.

EXAMPLES Example 1 RNA Synthesis

RNA encoding green fluorescent protein (“GFP”), NOVEPOETIN (“EPO”), elastin (“ELN”), tyrosinase (“TYR”), melanocortin-1-receptor (“MC1R”), HAS1, HAS2, HAS3, COL3A1, COL7A1, COL1A1, COL1A2, hTERT, Holly GFP, Fresno RFP, Blitzen Blue, RIBOSLICE gene-editing proteins, TALENs, Cas9, Oct4, Sox2, Klf4, c-Myc-2 (T58A), Lin28, IL2, IL6, IL15, IL22, BMP2, BMP7, BDNF, LIF, BMP6, IL15RA, FGF21, LIF, PTH, KRT5, KRT5-GFP, KRT14, KRT14-GFP, GDF15 and ESM1, and comprising various combinations of canonical and non-canonical nucleotides, was synthesized from DNA templates using the T7 High Yield RNA Synthesis Kit and the Vaccinia Capping System kit with mRNA Cap 2′-O-Methyltransferase (all from New England Biolabs, Inc.), according to the manufacturer's instructions and the present inventors' previously disclosed inventions (U.S. application Ser. No. 13/465,490 (now U.S. Pat. No. 8,497,124), International Application No. PCT/US12/67966, U.S. application Ser. No. 13/931,251, International Application No. PCT/US13/68118, and International Application No. PCT/US2015/013949, the contents of all of which are hereby incorporated by reference in their entirety) (Table 4). The RNA was then diluted with nuclease-free water to between 100 ng/μL and 2000 ng/μL. For certain experiments, an RNase inhibitor (Superase-In, Life Technologies Corporation) was added at a concentration of 1 μL/100 μg of RNA. RNA solutions were stored at room temperature, 4° C., −20° C. or −80° C. For reprogramming experiments, RNA encoding Oct4, Sox2, Klf4, c-Myc-2 (T58A), and Lin28 was mixed at a molar ratio of 3:1:1:1:1.

TABLE 4 RNA Synthesis Reaction ivT Template Nucleotides Volume/μL Yield/μg ELN A, 0.5 7dG, 0.4 5mU, 5mC 20 34.1 ELN A, 0.5 7dG, 0.4 5mU, 5mC 20 67.6 GFP A, 0.5 7dG, 0.4 5mU, 5mC 10 60.5 GFP A, 0.5 7dG, 0.4 5mU, 5hmC 10 25.5 GFP A, G, U, 5hmC 10 58.3 GFP A, 0.5 7dG, U, 5hmC 10 47.3 GFP A, 0.5 7dG, 0.4 5mU, 5cC 10 33.8 GFP A, G, U, 5hmC 15 30.3 GFP A, G, U, 5hmC 15 44.6 GFP A, G, U, 5hmC 15 24.7 TYR A, G, U, 5hmC 15 45.4 MC1R A, G, U, 5hmC 15 47.5 TYR A, G, U, C 20 67.0 TYR A, G, psU, C 20 93.7 TYR A, G, 0.4 5mU, C 20 85.7 TYR A, G, U, 5mC 20 73.4 TYR A, G, U, 5hmC 20 72.7 TYR A, 0.5 7dG, U, C 20 62.7 TYR A, G, psU, 5mC 20 116.3 TYR A, G, psU, 5hmC 20 102.4 TYR A, 0.5 7dG, psU, C 20 87.3 TYR A, G, 0.4 5mU, 5mC 20 106.5 TYR A, G, 0.4 5mU, 5hmC 20 85.0 TYR A, 0.5 7dG, 0.4 5mU, C 20 70.9 TYR A, 0.5 7dG, U, 5mC 20 88.5 TYR A, 0.5 7dG, U, 5hmC 20 59.1 TYR A, 0.5 7dG, psU, 5mC 20 7.8 TYR A, 0.5 7dG, psU, 5hmC 20 98.0 TYR A, 0.5 7dG, 0.4 5mU, 5mC 20 106.5 TYR A, 0.5 7dG, 0.4 5mU, 5hmC 20 82.3 HAS1 A, G, U, 5hmC 20 178.4 HAS2 A, G, U, 5hmC 20 59.3 HAS3 A, G, U, 5hmC 20 102.6 TYR A, G, 0.4 5mU, 5hmC 100 377.3 COL3A1 A, G, 0.4 5mU, 5hmC 20 108.3 COL7A1 A, G, 0.4 5mU, 5hmC 20 94.6 COL1A1 (20 μL) A, G, 0.4 5mU, 5hmC 20 114.0 COL1A2 (10 μL) A, G, 0.4 5mU, 5hmC 10 31.3 TYR A, G, 0.4 5mU, 5hmC 100 249.9 GFP A, G, 0.4 5mU, 5hmC 100 264.0 hTERT A, G, 0.4 5mU, 5hmC 100 349.2 GFP A, G, U, 5hC 20 81.7 GFP A, G, U, 0.5 5hC 20 65.4 GFP A, sG, U, C 20 34.7 GFP A, 0.5 sG, U,C 20 47.5 GFP A, G, 5hmU, C 20 22.1 GFP A, G, 0.5 5hmU, C 20 28.4 GFP A, G, 5cU, C 20 24.4 GFP A, G, 0.5 5cU, C 20 28.4 GFP A, G, 5moU, C 20 39.2 GFP A, G, 0.5 5moU, C 20 34.2 GFP A, G, U, C 20 42.0 GFP A, G, 5moU, C 20 53.8 GFP A, G, 5moU, 5hmC 20 101.5 GFP A, G, 0.4 5mU, 0.6 5moU, C 20 98.6 GFP A, G, 0.4 5mU, C 20 99.6 GFP A, G, U, 5mC 20 106.1 GFP A, G, U, C 20 85.7 GFP A, G, 5moU, C 100 398.4 hTERT A, G, 5moU, C 20 82.6 COL7A1 A, G, 5moU, C 20 34.9 COL7A1 A, G, 5moU, C 100 342.0 Holly GFP A, G, 5moU, C 20 36.7 Fresno RFP A, G, 5moU, C 20 72.0 Blitzen Blue A, G, 5moU, C 20 30.3 hTERT A, G, 5moU, C 20 49.6 Cas9 A, G, 5moU, C 20 31.6 EPO A, G, U, C 20 101.0 EPO A, G, 5moU, C 20 52.9 EPO A, G, psU, C 20 106.0 COL7A1 A, G, 5moU, C 20 80.2 Oct4 (SEQ ID NO: 8) A, G, 5moU, C 300 1925.5 Sox2 (SEQ ID NO: 9) A, G, 5moU, C 100 641.8 Klf4 (SEQ ID NO: 10) A, G, 5moU, C 100 739.0 c-Myc-2 (T58A) A, G, 5moU, C 100 574.0 Lin28 A, G, 5moU, C 100 556.0 IL2 A, G, 5moU, C 20 62.4 IL6 A, G, 5moU, C 20 22.2 IL15 A, G, 5moU, C 20 50.4 IL22 A, G, 5moU, C 20 63.6 BMP2 A, G, 5moU, C 20 83.2 BDNF A, G, 5moU, C 20 45.0 LIF A, G, 5moU, C 20 54.0 BMP6 A, G, 5moU, C 20 92.2 IL15RA A, G, 5moU, C 20 91.4 FGF21 A, G, 5moU, C 20 79.2 GFP A, G, 5moU, C 40 181.0 IL2 A, G, 5moU, C 30 99.4 IL6 A, G, 5moU, C 30 31.2 IL15 A, G, 5moU, C 30 89.8 IL22 A, G, 5moU, C 30 104.0 BDNF A, G, 5moU, C 30 95.9 BMP2 A, G, 5moU, C 30 112.0 LIF A, G, 5moU, C 30 116.0 PTH A, G, 5moU, C 30 88.4 EPO A, G, 5moU, C 30 83.3 KRT5 A, G, 5moU, C 15 66.6 KRT5-GFP A, G, 5moU, C 15 81.1 KRT14 A, G, 5moU, C 15 75.1 KRT14-GFP A, G, 5moU, C 15 90.4 GDF15 A, G, 5moU, C 15 71.1 A1AT TALEN L A, G, 5moU, C 15 56.4 A1AT TALEN R A, G, 5moU, C 15 57.3 A1AT RIBOSLICE L_A A, G, 5moU, C 15 74.3 A1AT RIBOSLICE L_B A, G, 5moU, C 15 56.4 A1AT RIBOSLICE R_A A, G, 5moU, C 15 60.3 A1AT RIBOSLICE R_B A, G, 5moU, C 15 35.7 COL7A1 exon 73 TALEN L A, G, 5moU, C 15 86.48 COL7A1 exon 73 TALEN R A, G, 5moU, C 15 83.66 COL7A1 exon 73 rs3L 50A A, G, 5moU, C 15 103.4 COL7A1 exon 73 rs3L 50B A, G, 5moU, C 15 112.8 COL7A1 exon 73 rs3R 50A A, G, 5moU, C 15 81.404 COL7A1 exon 73 rs3R 50B A, G, 5moU, C 15 78.02 COL7A1 exon 73 TALEN L A, G, 5moU, C 15 88.924 EA COL7A1 exon 73 TALEN L A, G, 5moU, C 15 75.2 Het COL7A1 exon 73 TALEN R A, G, 5moU, C 15 86.48 EA COL7A1 exon 73 TALEN R A, G, 5moU, C 15 62.98 Het COL7A1 exon 73 TALEN L A, G, 5moU, C 15 82.7 EA/Het COL7A1 exon 73 TALEN R A, G, 5moU, C 15 69.7 EA/Het HBB exon 1 TALEN L A, G, 5moU, C 15 112.8 HBB exon 1 TALEN R A, G, 5moU, C 15 108.1 PD-1 exon 1 TALEN L A, G, 5moU, C 15 95.88 PD-1 exon 1 TALEN R A, G, 5moU, C 15 101.52 ESM1 - transcript variant 1 A, G, 5moU, C 15 61 ESM1 - transcript variant 2 A, G, 5moU, C 15 66

“A” refers to adenosine-5′-triphosphate, “G” refers to guanosine-5′-triphosphate, “U” refers to uridine-5′-triphosphate, “C” refers to cytidine-5′-triphosphate, “7dG” refers to 7-deazaguanosine-5′-triphosphate, “sG” refers to thienoguanosine-5′-triphosphate, “5mC” refers to 5-methylcytidine-5′-triphosphate, “5hmC” refers to 5-hydroxymethylcytidine-5′-triphosphate, “5cC” refers to 5-carboxycytidine-5′-triphosphate, “5fC” refers to 5-formylcytidine-5′-triphosphate, “5hC” refers to 5-hydroxycytidine-5′-triphosphate, “psU” refers to 5-pseudouridine-5′-triphosphate, “5mU” refers to 5-methyluridine-5′-triphosphate, “5hmU” refers to 5-hydroxymethyluridine-5′-triphosphate, “5cU” refers to 5-carboxyuridine-5′-triphosphate, and “5moU” refers to 5-methoxyuridine-5′-triphosphate.

Example 2 Preparation of RNA-Transfection-Reagent Complexes

For each microgram of RNA, 1 μg RNA and 1 μL transfection reagent (LIPOFECTAMINE 3000, Life Technologies Corporation) were first diluted separately in complexation medium (Opti-MEM, Life Technologies Corporation or DMEM/F12+10 μg/mL insulin+5.5 μg/mL transferrin+6.7 ng/mL sodium selenite+2 μg/mL ethanolamine) to a total volume of between 5 μL and 100 μL each. Diluted RNA and transfection reagent were then mixed and incubated for 10 min at room temperature, according to the transfection reagent-manufacturer's instructions.

Example 3 Transfection of Cells with Synthetic RNA

Complexes were prepared according to Example 2, and were then added directly to cells in culture. For transfection in 6-well plates, between 10 μL and 250 μL of complexes were added to each well of the 6-well plate, which already contained 2 mL of transfection medium per well. Plates were shaken gently to distribute the complexes throughout the well. Cells were incubated with complexes for 4 hours to overnight, before replacing the medium with fresh transfection medium (2 mL/well). Alternatively, the medium was not replaced. Volumes were scaled for transfection in 24-well and 96-well plates.

Example 4 Toxicity of and Protein Translation from Synthetic RNA Containing Non-Canonical Nucleotides

Primary human fibroblasts were transfected according to Example 2, using RNA synthesized according to Example 1. Cells were fixed and stained 20-24 h after transfection using an antibody against Oct4. The relative toxicity of the RNA was determined by assessing cell density at the time of fixation.

Example 5 Delivery of Synthetic RNA to the Skin

The complexation reaction shown in Table 5 was prepared using RNA encoding green fluorescent protein (GFP) or collagen, type VII, alphal (COL7), synthesized according to Example 1. The concentration of the RNA stock solution was 500 μg/mL.

TABLE 5 RNA Complexation Reaction Volume RNA solution tube GFP or COL7 RNA  8 μL FactorPlex ™ complexation buffer 42 μL Transfection reagent solution tube LIPOFECTAMINE 3000 (LIFE TECHNOLOGIES)  4 μL FactorPlex ™ complexation buffer 46 μL

Each tube was mixed by pipetting, and the transfection reagent solution tube was incubated for 30 s at room temperature. The transfection reagent solution was then transferred to the RNA solution, and the contents were mixed by rapidly pipetting up and down 10 times. Following a 10 min incubation, dilutions were prepared according to

TABLE 6 Injection Solutions Complexation FactorPlex ™ RNA Site RNA Volume Volume amount 1 GFP 7.5 μL  22.5 μL   0.3 μg 2 GFP 15 μL 15 μL  0.6 μg 3 GFP 30 μL 0 μL 1.2 μg 4 COL7 30 μL 0 μL 1.2 μg

For each injection, the corresponding solution was drawn into a 3 cc insulin syringe with an 8 mm, 31 gauge needle (Becton, Dickinson and Company, Part Number: 328291) and air bubbles were removed. A clear field was selected on the left forearm of a healthy 33 year-old male human subject, and was disinfected with 70% isopropanol and allowed to dry. The needle was positioned at an angle of approximately 10° to the anterior (palmar) forearm with bevel facing up, and was inserted until the bevel was just covered. 30 μL of the RNA solution was injected intradermally over the course of approximately 10 sec. A distinct wheal appeared during the injection process. The needle was withdrawn, the wheal remained for approximately 1 minute, and no fluid escaped from the injection site. A total of 4 injections were performed according to Table 6, and all of the injections were performed between 11 and 28 minutes following the preparation of the RNA complexation reaction. No swelling, redness, or soreness occurred as a result of the injections. A small amount of bleeding occurred when the needle was removed from sites 2 and 4, resulting in the appearance of a small red spot at these sites.

The injection sites were imaged according to the schedule of Table 7, and every 24 hours thereafter for 6 days. Fluorescence images were acquired using an inverted microscope (Nikon Eclipse TS100) equipped with an EXFO X-Cite™ 120 fluorescence illumination system and the filter sets shown in Table 7. Fluorescence images were captured using a Sony NEX-7 digital camera (FIGS. 9-12).

TABLE 7 Measurement Parameters Site Time Image Type Exposure Time All  0 h Brightfield Automatic 1  0 h FITC 1/10 s 2  0 h FITC 1/10 s 3  0 h FITC 1/10 s 4  0 h FITC 1/10 s 1 12 h FITC 1/10 s 2 12 h FITC 1/10 s 3 12 h FITC 1/10 s 4 12 h FITC 1/10 s 1 12 h FITC 1/20 s 2 12 h FITC 1/20 s 3 12 h FITC 1/20 s 4 12 h FITC 1/20 s All 24 h Brightfield Automatic 1 24 h FITC 1/20 s 2 24 h FITC 1/20 s 3 24 h FITC 1/20 s 4 24 h FITC 1/20 s 1 24 h Cy3.5 ⅕ s 2 24 h Cy3.5 ⅕ s 3 24 h Cy3.5 ⅕ s 4 24 h Cy3.5 ⅕ s 1 24 h Cy3 ⅕ s 2 24 h Cy3 ⅕ s 3 24 h Cy3 ⅕ s 4 24 h Cy3 ⅕ s 1 36 h FITC 1/20 s 2 36 h FITC 1/20 s 3 36 h FITC 1/20 s 4 36 h FITC 1/20 s 1 48 h FITC 1/20 s 2 48 h FITC 1/20 s 3 48 h FITC 1/20 s 4 48 h FITC 1/20 s

An independent experiment was carried out using the 1.2 μg dose of GFP RNA, with similar results (FIG. 12).

TABLE 8 Filter Sets Image Type Filter Set Cy3 Chroma SP102V2 Cy3.5 Chroma SP103V2 FITC Chroma SP101

Example 6 Transfection of Human Keratinocytes with RNA Encoding NOVEPOETIN

RNA encoding NOVEPOETIN was synthesized according to Example 1 with three nucleotide combinations: 1) U, G, U, C, 2) U, G, 5moU, C, and 3) U, G, psU, C. Sub-confluent layers of primary human keratinocytes cultured in EpiLife medium were transfected in wells of a 6 well plate according to Example 3 with 1 μg of RNA per well. 12, 24, 36 and 48 h following transfection, 0.5 mL of medium was removed and 0.5 mL of fresh EpiLife medium was added to the plate. After the final medium sampling, the cells were harvested by trypsinization, and total RNA was isolated using the RNeasy mini kit (Qiagen). Genomic DNA was digested using DNase I and RNA was purified. Expression of interferon-3 and GAPDH was measured by RT-PCR (FIG. 5).

Example 7 Transfection of Human Cells with RNA Encoding hTERT

RNA encoding human telomerase reverse transcriptase (hTERT) was synthesized according to Example 1 with the following nucleotides: U, G, 5moU, C. A sub-confluent layer of primary human dermal fibroblasts cultured in DMEM+10% FBS were transfected in wells of a 24 well plate according to Example 3 with 0.25 μg of RNA per well. 12 h after transfection, cells were fixed and stained using a 1:50 dilution of rabbit anti-hTERT antibody (Millipore, Part Number: MABE14) (FIG. 16).

Example 8 High-Efficiency Gene Editing by Repeated Transfection with RIBOSLICE

Primary human fibroblasts were plated in 6-well plates coated with recombinant human fibronectin and recombinant human vitronectin (each diluted in DMEM/F12 to a concentration of 1 μg/mL, 1 mL/well, and incubated at room temperature for 1 h) at a density of 10,000 cells/well in transfection medium. The following day, the cells were transfected as in Example 2 with RNA synthesized according to Example 1. The following day cells in one of the wells were transfected a second time. Two days after the second transfection, the efficiency of gene editing was measured using a mutation-specific nuclease assay.

Example 9 Transfection of Cells with Synthetic RNA Containing Non-Canonical Nucleotides and DNA Encoding a Repair Template

For transfection in 6-well plates, 1 μg RNA encoding gene-editing proteins targeting exon 16 of the human APP gene, 1 μg single-stranded repair template DNA containing a PstI restriction site that was not present in the target cells, and 6 μL transfection reagent (LIPOFECTAMINE RNAiMAX, Life Technologies Corporation) were first diluted separately in complexation medium (Opti-MEM, Life Technologies Corporation) to a total volume of 120 μL. Diluted RNA, repair template, and transfection reagent were then mixed and incubated for 15 min at room temperature, according to the transfection reagent-manufacturer's instructions. Complexes were added to cells in culture. Approximately 120 μL of complexes were added to each well of a 6-well plate, which already contained 2 mL of transfection medium per well. Plates were shaken gently to distribute the complexes throughout the well. Cells were incubated with complexes for 4 hours to overnight, before replacing the medium with fresh transfection medium (2 mL/well). The next day, the medium was changed to DMEM+10% FBS. Two days after transfection, genomic DNA was isolated and purified. A region within the APP gene was amplified by PCR, and the amplified product was digested with PstI and analyzed by gel electrophoresis.

Example 10 In Vivo RIBOSLICE Safety Study

40 female NCr nu/nu mice were injected subcutaneously with 5×10⁶ MDA-MB-231 tumor cells in 50% Matrigel (BD Biosciences). Cell injection volume was 0.2 mL/mouse. The age of the mice at the start of the study was 8 to 12 weeks. A pair match was conducted, and animals were divided into 4 groups of 10 animals each when the tumors reached an average size of 100-150 mm³, and treatment was begun. Body weight was measured every day for the first 5 days, and then biweekly to the end of the study. Treatment consisted of RIBOSLICE BIRC5-1.2 complexed with a vehicle (LIPOFECTAMINE 2000, Life Technologies Corporation). To prepare the dosing solution for each group, 308 μL of complexation buffer (Opti-MEM, Life Technologies Corporation) was pipetted into each of two sterile, RNase-free 1.5 mL tubes. 22 μL of RIBOSLICE BIRC5-1.2 (500 ng/μL) was added to one of the two tubes, and the contents of the tube were mixed by pipetting. 22 μL of vehicle was added to the second tube. The contents of the second tube were mixed, and then transferred to the first tube, and mixed with the contents of the first tube by pipetting to form complexes. Complexes were incubated at room temperature for 10 min. During the incubation, syringes were loaded. Animals were injected either intravenously or intratumorally with a total dose of 1 μg RNA/animal in 60 μL total volume/animal. A total of 5 treatments were given, with injections performed every other day. Doses were not adjusted for body weight. Animals were followed for 17 days. No significant reduction in mean body weight was observed, demonstrating the in vivo safety of RIBOSLICE gene-editing RNA.

Example 11 Screening of Reagents for Delivery of Nucleic Acids to Cells

Delivery reagents including polyethyleneimine (PEI), various commercial lipid-based transfection reagents, a peptide-based transfection reagent (N-TER, Sigma-Aldrich Co. LLC.), and several lipid-based and sterol-based delivery reagents were screened for transfection efficiency and toxicity in vitro. Delivery reagents were complexed with RIBOSLICE BIRC5-1.2, and complexes were delivered to HeLa cells in culture. Toxicity was assessed by analyzing cell density 24 h after transfection. Transfection efficiency was assessed by analyzing morphological changes. The tested reagents exhibited a wide range of toxicities and transfection efficiencies. Reagents containing a higher proportion of ester bonds exhibited lower toxicities than reagents containing a lower proportion of ester bonds or no ester bonds.

Example 12 High-Concentration Liposomal RIBOSLICE

High-Concentration Liposomal RIBOSLICE was prepared by mixing 1 μg RNA at 500 ng/μL with 3 μL of complexation medium (Opti-MEM, Life Technologies Corporation), and 2.5 μL of transfection reagent (LIPOFECTAMINE 2000, Life Technologies Corporation) per μg of RNA with 2.5 μL of complexation medium. Diluted RNA and transfection reagent were then mixed and incubated for 10 min at room temperature to form High-Concentration Liposomal RIBOSLICE. Alternatively, a transfection reagent containing DOSPA or DOSPER is used.

Example 13 In Vivo RIBOSLICE Efficacy Study—Subcutaneous Glioma Model

40 female NCr nu/nu mice were injected subcutaneously with 1×10⁷ U-251 tumor cells. Cell injection volume was 0.2 mL/mouse. The age of the mice at the start of the study was 8 to 12 weeks. A pair match was conducted, and animals were divided into 4 groups of 10 animals each when the tumors reached an average size of 35-50 mm³, and treatment was begun. Body weight was measured every day for the first 5 days, and then biweekly to the end of the study. Caliper measurements were made biweekly, and tumor size was calculated. Treatment consisted of RIBOSLICE BIRC5-2.1 complexed with a vehicle (LIPOFECTAMINE 2000, Life Technologies Corporation). To prepare the dosing solution, 294 μL of complexation buffer (Opti-MEM, Life Technologies Corporation) was pipetted into a tube containing 196 μL of RIBOSLICE BIRC5-1.2 (500 ng/μL), and the contents of the tube were mixed by pipetting. 245 μL of complexation buffer was pipetted into a tube containing 245 μL of vehicle. The contents of the second tube were mixed, and then transferred to the first tube, and mixed with the contents of the first tube by pipetting to form complexes. Complexes were incubated at room temperature for 10 min. During the incubation, syringes were loaded. Animals were injected intratumorally with a total dose of either 2 μg or 5 μg RNA/animal in either 20 μL or 50 μL total volume/animal. A total of 5 treatments were given, with injections performed every other day. Doses were not adjusted for body weight. Animals were followed for 25 days.

Example 14 Liposome Formulation and Nucleic-Acid Encapsulation

Liposomes are prepared using the following formulation: 3.2 mg/mL N-(carbonyl-ethoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (MPEG2000-DSPE), 9.6 mg/mL fully hydrogenated phosphatidylcholine, 3.2 mg/mL cholesterol, 2 mg/mL ammonium sulfate, and histidine as a buffer. pH is controlled using sodium hydroxide and isotonicity is maintained using sucrose. To form liposomes, lipids are mixed in an organic solvent, dried, hydrated with agitation, and sized by extrusion through a polycarbonate filter with a mean pore size of 800 nm. Nucleic acids are encapsulated by combining 101 g of the liposome formulation per 1 μg of nucleic acid and incubating at room temperature for 5 minutes.

Example 15 Folate-Targeted Liposome Formulation

Liposomes are prepared using the following formulation: 3.2 mg/mL N-(carbonyl-ethoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (MPEG2000-DSPE), 9.6 mg/mL fully hydrogenated phosphatidylcholine, 3.2 mg/mL cholesterol, 2 mg/mL ammonium sulfate, and histidine as a buffer, with 0.27 mg/mL 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethylene glycol)-5000] (FA-MPEG5000-DSPE) added to the lipid mixture. pH is controlled using sodium hydroxide and isotonicity is maintained using sucrose. To form liposomes, lipids are mixed in an organic solvent, dried, hydrated with agitation, and sized by extrusion through a polycarbonate filter with a mean pore size of 800 nm. Nucleic acids are encapsulated by combining 101 g of the liposome formulation per 1 μg of nucleic acid and incubating at room temperature for 5 minutes.

Example 16 Therapy Comprising Liposomal Protein-Encoding RNA

Liposomes encapsulating synthetic RNA encoding a therapeutic protein, synthesized according to Example 1, are prepared according to Example 14 or Example 15. The liposomes are administered by injection or intravenous infusion.

Example 17 Generation of Elastin ivT-RNA Template

Total RNA was extracted from neonatal human dermal fibroblasts using the RNeasy mini kit (QIAGEN GmbH), according to the manufacturer's instructions. cDNA encoding human elastin was prepared using MonsterScript™ Reverse Transcriptase (Epicentre Biotechnologies) and the primer: AAAAAAACCGGT TCATTTTCTCTTCCGGCCAC (SEQ ID NO: 483). An in vitro transcription (ivT) template was prepared from the cDNA by PCR amplification of the elastin coding sequence (CDS) using the primers: F: AAAAAAGCTAGCATGGCGGGTCTGACG (SEQ ID NO: 484), and R: AAAAAAACCGGTTCATTTTCTCTTCCGGCCAC (SEQ ID NO: 485). The PCR product was then purified using agarose gel electrophoresis and the QIAquick Gel Extraction Kit (QIAGEN GmbH) and was cloned into a vector containing the human beta globin (HBB) 5′ and 3′ untranslated regions and a strong Kozak sequence. The vector was amplified, purified, and linearized prior to RNA synthesis.

Example 18 Generation of Tyrosinase ivT-RNA Template

Total RNA was extracted from human epidermal melanocytes using the RNeasy mini kit (QIAGEN GmbH), according to the manufacturer's instructions. cDNA encoding human tyrosinase was prepared using MonsterScript™ Reverse Transcriptase (Epicentre Biotechnologies). An in vitro transcription (ivT) template was prepared from the cDNA by PCR amplification of the tyrosinase coding sequence (CDS). The PCR product was then purified using agarose gel electrophoresis and the QIAquick Gel Extraction Kit (QIAGEN GmbH) and was cloned into a vector containing the human beta globin (HBB) 5′ and 3′ untranslated regions and a strong Kozak sequence. The vector was amplified, purified, and linearized prior to RNA synthesis.

Example 19 Synthesis of Tyrosinase RNA

RNA encoding human tyrosinase was synthesized according to Example 1, using the DNA template of Example 18 and the T7 High Yield RNA Synthesis Kit (New England Biolabs, Inc.), according to the manufacturer's instructions (Table 4). Samples of the RNA were analyzed by agarose gel electrophoresis to assess the quality of the RNA. The RNA was then diluted to 1 μg/μL. The RNA solution was stored at 4C.

Example 20 Increasing Melanin Production in Skin by Transdermal Injection via Syringe of RNA Encoding Tyrosinase

The RNA of Example 19 was loaded into a syringe and delivered by intradermal injection to the ventral forearm of a healthy 33 year-old male patient over the course of approximately 30 seconds.

Example 21 Increasing Melanin Production in Skin by Combined Delivery of RNA Encoding Tyrosinase and Electroporation

The area of skin treated in Example 20 was exposed to electrical pulses of between 10V and 155V and between approximately 10 milliseconds and approximately 1 second using a two-electrode array electrically connected to a capacitor. The patient reported a tingling sensation at all voltages and penetration depths. The treated area became darker after 24-48 hours. The experiment was repeated several times, with similar results.

Example 22 Increasing Melanin Production in Skin by Topical or Intradermal Application of RNA Encoding Tyrosinase

The RNA of Example 19 or the liposomes of Example 16 are applied directly to the skin, with or without disruption of the stratum corneum or injected intradermally or delivered by injection to the epidermis using a dose of one microgram or less per square centimeter. Optionally, an electric field is applied as in Example 21 or using a surface-contact patch to enhance delivery of the RNA.

Example 23 Increasing Elastin Production in Skin by Transdermal Delivery of RNA Encoding Elastin

RNA encoding elastin was prepared according to Example 1. The RNA is delivered as in Example 20, 21, or 22.

Example 24 Increasing Collagen Production in Skin by Transdermal Delivery of RNA Encoding Collagen

RNA encoding collagen was prepared according to Example 1. The RNA is delivered as in Example 20, 21, or 22.

Example 25 Anemia Therapy Comprising Delivery of RNA Encoding NOVEPOETIN

RNA encoding NOVEPOETIN was prepared according to Example 1. The RNA is delivered as in Example 20, 21, or 22.

Example 26 Increasing Production of Actin in Skeletal Muscle by Intramuscular Delivery of RNA Encoding Actin

RNA encoding actin is prepared according to Example 1. The RNA is delivered to the patient via intramuscular injection with or without the use of an electric field as in Example 20, 21, or 22.

Example 27 Wound Healing Treatment

RNA encoding basic fibroblast growth factor or IL22 is prepared according to Example 1. The RNA is delivered as in Example 20, 21, or 22.

Example 28 Anti-Scarring Treatment

RNA encoding collagenase is prepared according to Example 1. The RNA is delivered as in Example 20, 21, or 22.

Example 29 Production of Botulinum Toxin

RNA encoding botulinum toxin is prepared according to Example 1. The RNA is delivered as in Example 20, 21, or 22.

Example 30 Increasing Collagen Production in Skin Cells by Transfection with RNA Encoding Collagen I

RNA comprising the coding sequence of the human COL1A1 gene was synthesized according to Example 1. Primary human dermal fibroblasts were plated in wells of a 24-well plate, and were transfected according to Example 2. Between 24 and 72 hours after transfection, the cells were fixed and stained using an antibody targeting collagen I. Many extracellular deposits of collagen were visible in the transfected wells.

Example 31 Increasing Collagen Production in Skin Cells by Transfection with RNA Encoding Collagen VII

RNA comprising the coding sequence of the human COL7 gene was synthesized according to Example 1. Primary human dermal fibroblasts were plated in wells of a 24-well plate, and were transfected according to Example 2. Between 24 and 72 hours after transfection, the cells were fixed and stained using an antibody targeting collagen VII. Transfected cells exhibited high levels of collagen VII, compared to an un-transfected control.

Example 32 Increasing Collagen Production in Skin by Transdermal Injection Via Syringe of RNA Encoding Collagen I or Collagen VII

RNA comprising the coding sequence of the human COL1A1 gene or the human COL7 gene was synthesized according to Example 1. The RNA is loaded into a syringe and delivered to the dermis of a patient over the course of approximately 30 seconds or as in Example 20, 21, or 22.

Example 33 Increasing Collagen Production in Skin by Combined Delivery of RNA Encoding Collagen I or Collagen VII and Electroporation

The area of skin treated in Example 32 is exposed to electrical pulses of between 10V and 155V and between approximately 50 microseconds and approximately 1 second using a multi-electrode array electrically connected to a power source.

Example 34 Storage and Stability of Synthetic RNA Complexes

A complexation reaction using RNA encoding GFP was prepared according to Example 5. Following the 10 min incubation, the complexation reaction was divided into three equal parts, one of which was diluted 1:10 in FactorPlex™ complexation medium, one of which was diluted 1:10 in sterile, nuclease-free water, and one of which was left undiluted. Each of the three parts was then further divided into four equal parts, one of which was applied to primary human dermal fibroblasts according to Example 3, one of which was left at room temperature for six hours before applying to primary human dermal fibroblasts according to Example 3, one of which was placed at 4° C. for six hours before applying to primary human dermal fibroblasts according to Example 3, and one of which was snap frozen in liquid nitrogen and placed at −80° C. for six hours before applying to primary human dermal fibroblasts according to Example 3. The cells were imaged using a fluorescence microscope approximately 24 hours after the first transfection (FIG. 17). All wells contained GFP-positive cells, demonstrating that the synthetic RNA complexes were stable and maintained activity in all of the storage conditions tested.

Example 35: In Vivo Analysis of NOVECRIT

RNA encoding NOVEPOETIN was synthesized according to Example 1 with the nucleotide combination A, G, 5moU, C. In this Example, NOVECRIT was formulated with a lipid delivery vehicle, specifically LIPOFECTAMINE 3000. In this Example, NOVECRIT encoded NOVEPOETIN, a novel, high-stability erythropoiesis-stimulating agent.

A 15-day maximum tolerated dose (MTD) study was performed to evaluate safety (rat, n=62). In vivo toxicology and biodistribution, as well as pharmacodynamics and dose response and therapeutic effect, specifically on erythropoiesis, were evaluated. Furthermore, the immune response was monitored by analysis of cytokines in treated animals.

Sixty-nine, naïve, 8 week old male Sprague-Dawley rats (Rattus norvegicus) weighing 253 to 274 grams at receipt were used (HARLAN LABORATORIES). Animals were acclimated to the study room for at least seven days prior to dosing. On Day −2 all animals were shaved in the dorsal lumbar area and four intradermal sites (upper left, upper right, lower right and lower left) were designated on the dorsal back using a permanent marker. Sixty-four animals were randomly assigned to four treatment groups, with the remaining five animals serving as spares. Two animals were dropped from the study because of incomplete dosing.

The following study design was used (total dose volumes (μL) were constant):

Dose Dose Level Dose Conc. Vol. Group (μg) Route (μg/mL) (μL) Number of Males 1 0 Intradermal 0 50 4^(a) + 2^(b) + 2^(h) 2 0.25 Intradermal 5.0 50 4^(a) + 2^(b) + 2^(c) + 2^(d) + 2^(e) + 2^(f) + 2^(g) + 2^(h) 3 1.0 Intradermal 20 50 4^(a) + 2^(b) + 2^(c) + 2^(d) + 2^(e) + 2^(f) + 2^(g) + 2^(h) 4 4 × 1.0 μg Intradermal 20 50 4^(a) + 2^(b) + 3^(c) + 3^(d) + (4.0 μg 2^(e) + 2^(f) + 2^(g) + 2^(h) total) ID: ^(a)Toxicology animals necropsy on Day 15, TK animals with terminal tissue collections on Days 6^(b) and 24^(c) hours postdose, and on Days 3^(d), 4^(e), 6^(f), 8^(g), and 15^(h)

Blood was collected from the vena cava from anesthetized animals prior to necropsy or terminal tissue collection. Whenever possible, blood was collected via a single draw and then divided appropriately. Blood specimens for toxicokinetic analysis were collected from two animals per time point for Group 1 at 6 hours postdose and on Day 15. For Groups 2-4, blood specimens were collected at 6 and 24 hours postdose, and on Days 3, 4, 6, 8, and 15.

Blood specimens for hematology assessment were collected from all toxicology animals on Day 15 following an overnight fast, and all TK animals scheduled for terminal tissue collections at 6 and 24 hours postdose and on Days 8 and 15. Whole blood (1.3 mL) was deposited into K2EDTA tubes and analyzed using an Advia 120 automated analyzer.

Blood specimens for coagulation assessment were collected from all toxicology animals on Day 15 following an overnight fast. Coagulation specimens (1.8 mL) were collected into 3.2% sodium citrate tubes, processed according to standard procedures and analyzed using a STA Compact automated analyzer.

Blood specimens for serum chemistry assessments were collected from all toxicology animals on Day 15 following an overnight fast. Serum chemistry specimens (1 mL) were collected into serum separator tubes, processed according to standard procedures and analyzed using an AU680 analyzer.

Blood specimens for cytokine analysis were collected from animals scheduled for terminal tissue collection at 5 and 24 hours postdose and Day 8. Cytokine specimens (1 mL) were collected into K2EDTA tubes and processed to plasma according to standard procedures. TNFα, IL-6, and IFNα cytokine levels in plasma samples were studied. The study measured the production of TNFα, IL-6, and IFNα cytokines in plasma samples following dosing with the test article on Day 1 (6 hours post-dose), Day 2 (24 hours post-dose) and Day 8.

Study Outline for Cytokine Analysis Samples:

Dose NOVECRIT Concen- Dose Number Dose Level tration Volume of Rats Group (μg) (μg/mL) (μL) (Males) Endpoints 1 0 0 50 2^(b) Plasma TNFα, IL-6, 2 0.25 5.0 50 2^(b) + and IFNα cytokine 2^(c) + 2^(g) analysis 3 1.0 20 50 2^(b) + 2^(c) + 2^(g) 4 4 × 1.0 μg 20 50 2^(b) + (4.0 μg 2^(c) + 2^(g) total) ^(b)= plasma samples collected 6 hours post-dose, ^(c)= plasma samples collected 24 hours post-dose, ^(g)= plasma samples collected 8 days post-dose.

For TNFα analysis, plasma samples were analyzed using a commercially-available assay kit from R&D SYSTEMS (cat. no. RTA00). The rat TNFα ELISA is a solid phase enzyme-linked immunosorbent assay. The ELISA kit employs a TNFα-specific anti-rat monoclonal antibody for solid phase immobilization (pre-coated on the microtiter plate), and HRP conjugated to an anti-rat TNFα polyclonal antibody for detection. The reference standard provided with the kit is a recombinant rat TNFα. For each assay, plasma samples and standards were diluted with the diluent provided in each respective kit. For the TNFα ELISA, plasma samples were diluted 1:2. The standard curve consisted of six (6) serial 2-fold concentrations ranging from 800 to 12.5 pg/mL. Controls were reconstituted with diluent, and blanks contained diluent only.

Following sample, standard, control, or diluent addition (50 μL per well, each in duplicate), plates were incubated for 2 hours at room temperature. Following a wash step to remove unbound substances, HRP conjugate was added to each well (100 μL/well), and plates were incubated for 2 hours at room temperature. Following a second wash step, 100 μL/well TMB substrate was added to the plate. The plate was incubated for 30 minutes at room temperature, protected from light, to allow the color reaction to develop. The reaction was stopped following the addition of HCl Stop Solution (100 μL/well). The optical density was read on a SpectraMax 340 (MOLECULAR DEVICES) plate reader at 450 nm, within 30 minutes following addition of Stop Solution. The intensity of the color measured was in proportion to the amount of rat TNFα bound in the initial step. A standard curve was generated for each assay plate, and test sample TNFα concentrations were determined by interpolation of absorbance A₄₅₀ values from the standard curve and dilution factor. The assay range for the TNFα kit is 12.5-800 pg/mL, with a minimum detectable concentration of less than 10 pg/mL (with the 1:2 minimum required dilution for plasma samples).

For IL-6 analysis, plasma IL-6 samples were analyzed using a commercially-available assay kit from R&D SYSTEMS (cat. no. R6000B—IL-6). The rat IL-6 ELISA is a solid phase enzyme-linked immunosorbent assay. The ELISA kit employs anti-rat IL-6 monoclonal antibody for solid phase immobilization (pre-coated on the microtiter plate), and HRP conjugated to IL-6-specific anti-rat polyclonal antibody for detection. The reference standard provided with the kit is a recombinant rat IL-6. Plasma samples were used undiluted (as provided), and standards were diluted with the diluent provided in the kit. The standard curve consisted of six (6) serial 2-fold concentrations ranging from 4,000 to 62.5 pg/mL. Controls were reconstituted with diluent, and blanks contained diluent only. Following sample, standard, control, or diluent addition (50 μL per well, each in duplicate), plates were incubated for 2 hours at room temperature. Following a wash step to remove unbound substances, HRP conjugate was added to each well (100 μL/well), and plates were incubated for 2 hours at room temperature. Following a second wash step, 100 μL/well TMB substrate was added to the plate. The plate was incubated for 30 minutes at room temperature, protected from light, to allow the color reaction to develop. The reaction was stopped following the addition of HCl Stop Solution (100 μL/well). The optical density was read on a SpectraMax 340 (MOLECULAR DEVICES) plate reader at 450 nm, within 30 minutes following addition of Stop Solution. The intensity of the color measured was in proportion to the amount of rat IL-6 bound in the initial step. A standard curve was generated for each assay plate, and test sample IL-6 concentrations were determined by interpolation of absorbance A450 values from the standard curve and dilution factor. The assay range for the IL-6 kit is 62.5-4,000 pg/mL, with a minimum detectable concentration of less than 21 pg/mL.

For IFNα analysis, plasma IFNα samples were analyzed using a commercially-available assay kit from NOVATEINBIO (cat. no. BG-RAT11380). The ELISA is a solid phase enzyme-linked immunosorbent assay. The ELISA kit employs anti-rat IFNα monoclonal antibody for solid phase immobilization (pre-coated on the microtiter plate), and HRP conjugated antibody specific for IFNα for detection. Plasma samples were diluted 1:4, and the standards were used as provided in the kit. Standards consisted of six (6) serial 2-fold concentrations ranging from 100 to 3.1 pg/mL. Blanks contained diluent only. Following sample, standard, or diluent addition (50 μL per well, each in duplicate), HRP conjugate was added to each well (100 μL/well), and plates were incubated for 1 hours at 37° C. Following a wash step, 50 μL/well each of Chromogen Solution A and Chromogen Solution B were added to the plate. The plate was incubated for 15 minutes at 37° C., protected from light, to allow the color reaction to develop. The reaction was stopped following the addition of Stop Solution (50 μL/well). The optical density was read on a SpectraMax 340 (MOLECULAR DEVICES) plate reader at 450 nm. The intensity of the color measured was in proportion to the amount of rat IFNα bound in the initial step. A standard curve was generated for each assay plate, and test IFNα concentrations were determined by interpolation of absorbance A450 values from the standard curve and dilution factor. The assay range for the IFNα kit is 3.1-100 pg/mL, with a minimum detectable concentration of less than 1 pg/mL (4 pg/mL, with the 1:4 dilution required for plasma samples).

The results of this study were, inter alia, that NOVECRIT was not detected at the injection site 24 h after dosing (as assayed by RT-PCR). More specifically, NOVECRIT was not detected in any of the following samples (RT-PCR): serum (6 h, 24 h, 48 h and 72 h after dosing), liver (6 h and 24 h after dosing), and kidney (6 h and 24 h after dosing). Further, positive controls (spiked with NOVECRIT) yielded a robust signal in all tissues (as assayed RT-PCR). FIG. 18 depicts single administration of NOVECRIT induced a rapid increase and sustained level of NOVEPOIETIN in serum. The Y axis shows concentration of NOVEPOIETIN protein (mU/mL). This suggests, without wishing to be bound by theory, that the studied RNA therapeutic is able to provide therapeutically important pharmacodynamic properties. In fact, this PD behavior is vastly improved relative to wild type EPO (which is known in the art to have a half-life of about 4-12 hours).

Furthermore, serum spiked with NOVECRIT showed near-instant degradation of the RNA (as assayed by RT-PCR). Without wishing to be bound by theory, this data indicates that the present RNA therapeutics are safe and have little chance of toxicity limitations, for example, liver or kidney toxicities.

FIG. 19 depicts a single administration of NOVECRIT stimulated erythropoiesis, yielding elevated hematocrit for at least 14 days. The left panel shows % hematocrit on the Y axis, while the right panel shows % reticulocytes. Accordingly, the studied RNA therapeutic is fully functional.

With respect to cytokines, IL-6 and TNFα cytokine levels were below assay detection thresholds for all animals at each of the timepoints evaluated in this study. Low levels of IFN-α were only detectable in the Day 8 samples from animals in Groups 3 and 4. FIG. 20 depicts a table summarizing TNFα, IL-6, and IFNα cytokine levels in plasma samples collected from a maximum tolerated dose of NOVECRIT in male Sprague Dawley rats. Without wishing to be bound by theory, this data indicates that the present RNA therapeutics do not stimulate an unfavorable immunogenicity which has limited the therapeutic utility of certain RNA therapeutics.

Example 36: Gene Editing of the COL7A1 Gene

The present RNA-based gene editing approaches were applied to the COL7A1 gene. This gene is of interest because, inter alia, it is frequently involved in dystrophic epidermolysis bullosa. FIG. 21 depicts a SURVEYOR assay using the DNA of primary adult human dermal fibroblasts transfected with RNA TALENs targeting the sequence TGAGCAGAAGTGGCTCAGTG (SEQ ID NO: 467) and TGGCTGTACAGCTACACCCC (SEQ ID NO: 468), located within the COL7A1 gene. The bands present in the +RNA lane indicate editing of a region of the gene that is frequently involved in dystrophic epidermolysis bullosa. FIG. 22 depicts another SURVEYOR assay using the DNA of primary adult human dermal fibroblasts transfected with RNA TALENs, now targeting the sequence TTCCACTCCTGCAGGGCCCC (SEQ ID NO: 469) and TCGCCCTTCAGCCCGCGTTC (SEQ ID NO: 470), located within the COL7A1 gene. The bands present in the +RNA lane indicate editing of a region of the gene that is frequently involved in dystrophic epidermolysis bullosa. This data points to, among others, a gene editing approach to the treatment of certain genetic disorders such as dystrophic epidermolysis bullosa.

Example 37: Gene-Editing of the MYC Gene Using a Synthetic RNA with Non-Canonical Nucleotides

Experiments were conducted with in vitro transcribed synthetic RNA molecules containing non-canonical nucleotides and encoding gene-editing proteins. The immunogenicity and the gene-editing efficiency of in vitro transcribed synthetic RNA molecules having (1) only pseudouridine (psU) as a non-canonical nucleotide; (2) only 5-methylcytidine (5mC) as a non-canonical nucleotide; and (3) both of pseudouridine and 5-methylcytidine as non-canonical nucleotides was evaluated (as well as controls).

Specifically, RNA containing the following nucleotide combinations: (i) A,G,U,C, (ii) A,G,psU,C, (iii) A,G,U,5mC, and (iv) A,G,psU,5mC, and encoding TALEN pairs targeting the following DNA sequences, which can be found within the MYC gene: TCGGCCGCCGCCAAGCTCGT (SEQ ID NO: 474) and TGCGCGCAGCCTGGTAGGAG (SEQ ID NO: 475), were synthesized according to the methods described herein. Human dermal fibroblasts (MA001SK) were plated in 6-well and 24-well tissue culture plates in DMEM with 10% FBS at 100,000 and 10,000 cells per well, respectively. The next day, the cells were transfected in the 6-well plate with 2 μg of RNA (1 μg for each component of the TALEN pair) and the cells were transfected in the 24-well plate with 0.2 μg of RNA (0.1 μg for each component of the TALEN pair) according to the methods described herein. 24 hours after transfection, the total RNA from the cells in the 24-well culture plate was isolated using an RNeasy Mini Kit (74106; QIAGEN), including isolating the total RNA from a sample of cells that had not been transfected with RNA (negative control; “Neg.” in FIG. 23). The genomic DNA was removed by a 15 minute digestion with DNase I (RNase-Free) (M0303L; NEW ENGLAND BIOLABS) and the reaction purified using an RNeasy Mini Kit. 1 μL of total RNA was used to assess gene expression by real-time RT-PCR using TAQMAN gene-expression assays (APPLIED BIOSYSTEMS) designed to detect expression of the immunogenicity markers TLR3, IFIT1, and IFIT2 (FIG. 23). The data were normalized to both the positive experimental control sample (“A,G,U,C”) and to a loading control (GAPDH).

48 hours after transfection, the genomic DNA was isolated from the cells in the 6-well culture plate using a DNeasy Blood and Tissue Kit (69506; QIAGEN), including from a sample of cells that had not been transfected with RNA (negative control, “Neg.” in FIG. 24). A 970 bp region of the MYC gene surrounding the predicted TALEN cut location was amplified using a 35 cycle 2-step PCR reaction containing the following primers: TAACTCAAGACTGCCTCCCGCTTT (SEQ ID NO: 476) and AGCCCAAGGTTTCAGAGGTGATGA (SEQ ID NO: 477). 160 ng was hybridized in 5 μL of amplified sequence from RNA-treated cells to 160 ng in 5 μL of amplified sequences from untreated MA001SK cells by mixing the two sequences with 0.5 μL of 1M KCl and 0.5 μL of 25 mM MgCl₂ and running the following program in a thermocycler: 95° C. for 10 minutes; 95° C. to 85° C. at 0.625 C/s; 85° C. to 25° C. at 0.125 C/s. The SURVEYOR assay was performed by adding 0.5 μL of SURVEYOR nuclease and 0.5 μL of Enhancer from the SURVEYOR Mutation Detection Kit (7060201; INTEGRATED DNA TECHNOLOGIES) to the hybridized product, mixing, and incubating at 42° C. for 25 minutes. The protocol above was also used to process the positive control DNA sample provided with the SURVEYOR Mutation Detection Kit as a positive experimental control for the SURVEYOR Assay (“Assay Pos.” in FIG. 24). Samples were analyzed by agarose gel electrophoresis (FIG. 24). For each sample, gene-editing efficiency was calculated as a ratio of the intensity of the digested bands (indicated by “*” in FIG. 24) to that of the undigested band.

As shown in FIG. 23 below, the samples from cells transfected with the positive control RNA (A,G,U,C), and the samples from cells transfected with RNA containing either pseudouridine or 5-methylcytidine exhibited upregulation of all three of the immunogenicity markers TLR3, IFIT1, and IFIT2. The sample from cells transfected with RNA containing both pseudouridine and 5-methylcytidine exhibited negligible upregulation of the immunogenicity markers (less than 0.01-fold of the positive control), demonstrating that in vitro transcribed synthetic RNA with both pseudouridine and 5-methylcytidine and encoding a gene-editing protein can evade detection by the innate-immune system of mammalian cells.

Further, as shown in FIG. 24 below, the sample from cells transfected with RNA containing both pseudouridine and 5-methylcytidine exhibited highly efficient gene editing (41.7%), which was greater than the efficiency exhibited by samples from cells transfected with RNA containing pseudouridine alone (35.2%), demonstrating that in vitro transcribed synthetic RNA comprising both pseudouridine and 5-methylcytidine and encoding a gene-editing protein can both (i) gene-edit mammalian cells at high efficiency, and (ii) gene-edit mammalian cells at higher efficiency than in vitro transcribed synthetic RNA comprising pseudouridine and not comprising 5-methylcytidine.

Example 38: COL7A1 Gene Editing and Repair in Human Cells

RNA encoding gene editing proteins targeting the following sequences in the COL7A1 gene was synthesized according to Example 1: TGAGCAGAAGTGGCTCAGTG (SEQ ID NO: 473) and TGGCTGTACAGCTACACCCC (SEQ ID NO: 468) (see also table below).

RNA Synthesis

Reaction ivT Template Nucleotides Volume/μL Yield/μg COL7A1 TALEN 1L A, G, 5moU, C 20 120.528 COL7A1 TALEN 1R A, G, 5moU, C 20 120.204 COL7A1 TALEN 1L A, G, 5moU, C 15 81.94 COL7A1 TALEN 1R A, G, 5moU, C 15 61.88

50,000 primary human epidermal keratinocytes (HEKn, Gibco) were plated in wells of 6-well plates in EpiLife+Supplement S7. The next day, cells were transfected according to Example 3 with 1 μg of RNA encoding each component of the gene editing pair and 2 μg of a single-stranded DNA repair template having a length of 60, 70, 80, 90 or 100 nucleotides (“nt”). 48 hours after transfection, genomic DNA was purified. A segment of the COL7A1 gene was amplified using the primers GCATCTGCCCTGCGGGAGATC (SEQ ID NO: 478) and CCACGTTCTCCTTTCTCTCCCCGTTC (SEQ ID NO: 479), which produce a 535 bp amplicon. The efficiency of gene editing was assessed using T7 Endonuclease I (“T7E1”, New England Biolabs) according to the manufacturer's instructions. Bands of approximately 385 bp and 150 bp indicate successful gene editing. FIG. 25 and FIG. 29 show the result of digestion with T7EI, analyzed by agarose gel electrophoresis. FIG. 27 and FIG. 29 show the result of digestion with MluI-HF, analyzed by agarose gel electrophoresis. Because the repair template contains the sequence ACGCGT (SEQ ID NO: 480), digestion of the amplified product with MluI-HF (New England Biolabs) produces bands of approximately 385 bp and 150 bp in the case of successful gene repair.

RNA encoding gene editing proteins targeting the following sequences in the COL7A1 gene was synthesized according to Example 1: TGAGCAGAAGTGGCTCAGTG (SEQ ID NO: 473) and TGGCTGTACAGCTACACCCC (SEQ ID NO: 468). 50,000 primary human epidermal keratinocytes (HEKn, Gibco) were plated in wells of 6-well plates in EpiLife+Supplement S7. The next day, cells were transfected according to Example 3 with 1 μg of RNA encoding each component of the gene editing pair and 1-4 μg of an 80 nucleotide single-stranded DNA repair template. 48 hours after transfection, genomic DNA was purified. A segment of the COL7A1 gene was amplified using the primers GCATCTGCCCTGCGGGAGATC (SEQ ID NO: 481) and CCACGTTCTCCTTTCTCTCCCCGTTC (SEQ ID NO: 482), which produce a 535 bp amplicon. The efficiency of gene editing was assessed using T7 Endonuclease I (“T7E1”, New England Biolabs) according to the manufacturer's instructions. Bands of approximately 385 bp and 150 bp indicate successful gene editing. FIG. 26 show the result of digestion with T7EI, analyzed by agarose gel electrophoresis. FIG. 28 show the result of digestion with MluI-HF, analyzed by agarose gel electrophoresis. Because the repair template contains the sequence ACGCGT (SEQ ID NO: 480), digestion of the amplified product with MluI-HF (New England Biolabs) produces bands of approximately 385 bp and 150 bp in the case of successful gene repair.

Example 39: Expression of BMP7 Variants in Human Cells

RNA encoding wild type BMP7 and RNA encoding variants of BMP7 was synthesized according to Example 1 (see also table below).

RNA Synthesis

Reaction ivT Template Nucleotides Volume/μL Yield/μg BMP7 Wild Type A, G, 5moU, C 15 125.8 BMP7 Variant A A, G, 5moU, C 15 120.36 BMP7 Variant B A, G, 5moU, C 15 143.14 BMP7 Variant C A, G, 5moU, C 15 106.42

50,000 primary human dermal fibroblasts (MA001SK, Factor Bioscience) or 100,000 primary human epidermal keratinocytes (HEKn, Gibco) were plated in DMEM+10% FBS or EpiLife+Supplement S7, respectively. Cells were transfected according to Example 3 with 1 μg of RNA encoding wild type BMP7 or a variant thereof. 24 hours after transfection, the medium was sampled and secreted BMP7 levels were measured with a human BMP7 ELISA kit (ab99985, Abcam) using medium diluted 10-fold according to the manufacturer's instructions. Secreted BMP7 levels were determined by measuring 450 nm absorbance using a microplate reader (EMax Plus, Molecular Devices). Secreted BMP7 levels are shown in FIG. 30.

Example 40: Expression of Parathyroid Hormone (PTH) in Human Cells

RNA encoding PTH was synthesized according to Example 1 (see also table below).

RNA Synthesis

Reaction ivT Template Nucleotides Volume/μL Yield/μg PTH A, G, 5moU, C 15 51.68

100,000 human epidermal keratinocytes (HEKn, Gibco) were plated in EpiLife+Supplement S7. Cells were transfected according to Example 3 with 1 μg of RNA encoding PTH. 24 hours after transfection, the medium was sampled and secreted PTH levels were measured using a human PTH ELISA kit (EIA-PTH-1, Ray Biotech) according the manufacturer's instructions. Secreted PTH levels were determined by measuring 450 nm absorbance using a microplate reader (EMax Plus, Molecular Devices). Secreted PTH levels are shown in FIG. 31.

Example 41: Intradermal, Subcutaneous, Rectal and Nasal Administration of NOVECRIT for the Treatment of Anemia

A repeat dose toxicity study of Novecrit was conducted for the treatment of anemia. Specifically, 8-10 weeks old male Sprague Dawley rats were administered with Novecrit via intradermal, subcutaneous, rectal, or nasal routes once per day on days 1, 8, and 15. The animals were assigned to groups and treated as indicated in the table below:

Dose Dose Number of Group Test Concentration Volume Animals Group Color Article Dose Route Dose Level (μg) (μg/mL) (μL) Males 1 White Control Intradermal 0.0 0.0 50 3^(a) + 8^(b) 4X 2 Yellow Novecrit Intradermal (0.25 μg/ 5.0 50 3^(a) + 8^(b) injection; 1.0 μg total) 3 Green Novecrit Subcutaneous 4.0 20.0 200 3^(a) + 8^(b) 4 Blue Novecrit Rectal 4.0 20.0 200 3^(a) + 8^(b) 5 Red Novecrit Nasal 4.0 20.0 200 3^(a) + 8^(b) Note: Total dose volume (μL/per animal) are constant ^(a)Toxicity animals (also called main study animals), necropsy on Day 44 ^(b)TK animals, euthanized as n = 2/time point on Days 2, 16, 23 and 44

More particularly, Group 1 was dosed via intradermal injection. Each dose was administered in four intradermal injections of 50 μL/injection for a total of 200 uL per animal. Injections were carried out on previously marked sites near the midline of the dorsal lumbar area (upper left, upper right, lower left and lower right quadrants). Group 2 was dosed four intradermal injections at 0.25 μg each (1.0 μg total) into previously marked sites near the midline of the dorsal lumbar area (upper left, upper right, lower left and lower right quadrants). Group 3 was dosed by subcutaneous injections into an area of the back located in the lower dorsal thoracic/lumbar region. Group 4 was dosed by rectal administration. The animal was manually restrained and the abdomen of each rat was manually palpated to remove any fecal matter. If deemed necessary, the rectum was lavaged with up to 2 ml of saline for enema followed by manual palpation of the abdomen to remove the fecal matter, if any. Novecrit was drawn into a syringe and an appropriately sized gavage needle with a rounded tip (ball) was attached to the syringe. A lubricant jelly was applied to the insertion device to aid with insertion; it was advanced approximately 1 cm into the lumen of the colon and the Novecrit instilled. The rat was then maintained in a head-down position for approximately 20-30 seconds to limit the expulsion of solution. Group 5 was dosed via nasal route. The animal was anesthetized (per SNBL USA SOP). The animal was laid on its back with the head elevated. The dose was dispensed slowly into the nares. Approximately half the dose volume was administered to one nare. The remaining dose volume was administered to another nare. The first dose was administered on Day 1, and the last dose on Day 15.

The rats were clinically monitored including their food consumption and body weight. In addition, blood was collected for hematology, coagulation, and serum chemistry analysis as indicated below:

Toxicity Group Specimen collection frequency Time Point Hematology Coagulation Serum Chemistry Day 44 1X 1X 1X (n = 3/Group) (n = 3/Group (n = 3/Group) X = Number of times procedure performed

Analysis was done on the toxicokinetic group as follows:

In-text Table 3: Toxicokinetic Group Specimen collection frequency Time Point Hematology^(a) TK Cytokines Acclimation 1X — — (n = 8/Group) Day 2 — 1X 1X (n = 2/Group, (n = 2/Group, 24 hours postdose) 24 hours postdose) Day 8 Pre-dose — — (n = 6/Group) Day 15 Pre-dose — — (n = 6/Group) Day 16 — 1X 1X (n = 2/Group, (n = 2/Group, 24 hours postdose) 24 hours postdose) Day 22 1X — — (n = 4/Group) Day 23 — 1X 1X (n = 2/Group) (n = 2/Group) Day 29 1X — — (n = 2/Group) Day 36 1X — — (n = 2/Group) Day 43 1X — — (n = 2/Group) Day 44 — 1X 1X (n = 2/Group) (n = 2/Group) X = Number of times procedure performed TK = toxicokinetics

Terminal necropsy for toxicity animals occurred on Day 44. TK animals were euthanized on Days 2, 16, 23, and 44. Pathological analysis was conducted on the animals.

As shown in FIG. 32, administration of NOVECRIT stimulated erythropoiesis resulting in elevated hematocrit for at least 14 days in all four study groups compared to control.

Example 42: Intradermal Administration of RNA Encoding BMP7 Variants for the Treatment of Diabetic Nephropathy

A ZDSD rat model was utilized to study the effects of RNAs encoding BMP7 variants for the prevention and treatment of diabetic nephropathy. Specifically, ZDSD rats were treated with RNAs encoding BMP7 variants administered intradermally. A schematic of the study design is provided in FIG. 33. The animals were assigned to groups and treated as indicated in the table below:

Dose Dose Route Group No. of Dose Level Conc. Volume of Number Treatment Animals (μg) (μg/ml) (ml/kg) Frequency Admin. 1 Vehicle 24 0 0 0.1 2x/wk I.D. 2 FTB-F1 (wt 6 1 5 0.1 2x/wk I.D. BMP7) 3 FTB-F2 (BMP7 6 1 5 0.1 2x/wk I.D. variant A) 4 FTB-F3 (BMP7 6 1 5 0.1 2x/wk I.D. variant B) 5 Lisinopril 6 Standard Standard 5 Continuous Diet  6^(a) Vehicle 20 0 0 0.2/rat 3x/wk I.D.  7^(a) FTB-F2 10 2 10  0.2/rat 3x/wk I.D. (BMP7 variant A) ^(a)Group 1 animals at the end of the “Prevention” arm were randomized into Groups 6 and 7 (n = 20 or 10 each, respectively) to define the “Treatment” arm. The remaining 6 animals from Group 1 were euthanized for kidney collection and blood draw at the end of the “Prevention” arm.

To study the effect of the RNAs on prevention of diabetic nephropathy, animals were delivered from the barrier (PCO-5638) to the vivarium (PCO-Rm C) and allowed 3 days for acclimation. All animals were placed on 5SCA diabetogenic diet for 3 weeks (weekly body weight measurements were taken). Animals were then returned to regular 5008 diet for the duration of the study (weekly body weight measurements were taken). After 2 weeks on 5008 diet, body weight measurement, blood draw (500 ul) and a 24 hour baseline urine collection (urinary albumin and creatinine measurements) were performed on all animals. 36 rats were randomized to groups 1, 2 and 3 based on body weight, glucose and urinary albumin. Groups 1, 2, 3 and 4 received either vehicle or test article (i.d.) via intradermal administration (2×/week, Mon and Thur). Group 5 was administered Lisinopril admixed in the 5008 diet (food consumption will begin on this group of animals). Blood draws (500 ul, tail vein) were taken from Groups 2, 3 and 4, 24 hours after each dose for the first 2 weeks and then 1×/week thereafter. After 4 weeks of dosing, blood sample via tail vein (500 ul) was taken from all animals (Groups 1-5). At the end of 8 weeks of dosing, a 24 hour urine collection was performed (urinary albumin and creatinine measurements) together with a tail vein (500 ul) blood sample from Group 1 (n=18 going into the Treatment arm). Results for urine volume, creatinine, and albumin is shown in FIG. 35. As shown in FIG. 35, treatment with RNA encoding BMP7 Variant A resulted in reduced levels of urine albumin in rats afflicted with diabetic nephropathy compared to the control, indicating superior kidney function in the treated animals compared to the control animals (placebo group). To terminate the study, all animals were euthanized with CO₂ asphyxiation and a blood draw was performed via cardiac puncture to collect serum for BUN, creatinine and glucose, and plasma for compound exposure and biomarker analysis. Both kidneys were dissected and fixed for histology. FIG. 34 shows the serum level of BMP7 protein in Groups 1, 2, 3, and 4. Results indicate that the serum levels of BMP7 protein in these groups were as follows: Group 4, Group 3>Group 2, Group 1.

A study was also conducted to analyze the effect of the RNAs encoding BMP7 variants on treating diabetic nephropathy. Specifically, the vehicle animals from Group 1 (Prevention arm, n=18) were randomized based on body weight, glucose and urine albumin (Groups 6, n=20; 7, n=10). Animals received either Vehicle or BMP7 variant-encoding RNAs (intradermal, 2×/week, Mon and Thur), groups 6 and 7, respectively. After 4 weeks of treatment, animals were euthanized with CO₂ asphyxiation and a blood draw will be performed via cardiac puncture to collect serum for BUN, creatinine and glucose, and plasma for compound exposure and biomarker analysis. Both kidneys were dissected and fixed for histology. As shown in FIG. 36, treatment with RNA encoding BMP7 Variant A significantly decreased urine albumin level in rats afflicted with diabetic nephropathy (p=0.0015), indicating superior kidney function in the treated animals. In addition, the treatment group showed significantly lower kidney weight at termination (p=0.018), and significantly lower serum creatinine at termination (p=0.016). These results suggested that BMP7 variant-encoding RNA effectively treated diabetic nephropathy and promoted superior kidney function compared to the control animals (placebo group).

An illustrative study protocol is provided below:

Week Day Procedure 15 weeks −7 Animals arrive in Room C from the barrier old 0 BW*, Stat Strip, and Place animals on 5SCA for 3 wks 1 7 BW*, Stat Strip 2 14 BW*, Stat Strip 3 21 BW*, Stat Strip, put animals on 5008 4 28 BW*, Stat Strip 34 BW*, Tail vein blood draw and randomize for Prevention arm (Grps 1, 2, 3, 4, 5) 5 35 Begin 24 hr urine collection 36 Collect 24 hr urine 39 BW* Intradermal dosing Grps 1, 2, 3 and 4, and put Grp 5 on admixed 5008 diet Begin food consumption on group 5 40 Collect 24 blood sample for exposure (gps 2, 3, & 4) 42 Dosing 43 Collect 24 blood sample for exposure (gps 2, 3, & 4)  6 (1 P) 46 BW*, Dosing Food consumption group 5 47 Collect 24 blood sample for exposure (gps 2, 3, & 4) 49 Dosing 50 Collect 24 blood sample for exposure (gps 2, 3, & 4)  7 (2 P) 53 BW*, Dosing Food consumption group 5 54 Collect 24 blood sample for exposure (gps 2, 3, & 4) 56 Dosing  8 (3 P) 60 BW*, Dosing Food consumption group 5 61 Collect 24 blood sample for exposure (gps 2, 3, & 4) 63 Dosing  9 (4 P) 67 BW*, Dosing Food consumption group 5 Blood draw (tail vein) 68 Blood draw (all groups) Collect 24 blood sample for exposure (gps 2, 3, & 4) 70 Dosing 10 (5 P) 74 BW*, Dosing Food consumption group 5 75 Collect 24 blood sample for exposure (gps 2, 3, & 4) 77 Dosing 11 (6 P) 81 BW*, Dosing Food consumption group 5 Collect 24 blood sample for exposure (gps 2, 3, & 4) 84 Dosing 12 (7 P) 88 BW*, Dosing Food consumption group 5 89 Collect 24 blood sample for exposure (gps 2, 3, & 4) 91 Dosing 13 (8 P) 95 BW*, dosing Food consumption group 5 Blood draw (tail vein) (all groups) 96 Begin 24 hr urine collection 97 Collect 24 hr urine Terminate Groups 1 (n = 6), 2, 3, 4 and 5 cardiac puncture blood draw, and collect kidneys, weigh and fix. 98 BW* and randomize (BW*, glucose, albumin) Group 1 (n = 18) rats into Groups 6, 7 and 8 Dose Food Consumption group 8 103 Dose 14 (1 T) 106 BW*, Dosing Food Consumption group 8 110 Dosing 15 (2 T) 113 BW*, Dosing Food Consumption group 8 117 Dosing 16 (3 T) 120 BW*, Dosing Food Consumption group 8 124 Dosing 17 (4T)  127 BW*, Terminate, cardiac puncture blood draw, and collect kidneys, weigh and fix. *BW: body weight measurement

In an illustrative study, the protocol was amended from above towards the end of the study as follows:

Week [Treatment Arm] Day BW Begin 24 hr urine collection 81 Collect 24 hr urine BW, Blood draw Randomize (BW, glucose, albumin) rats into Groups 6 and 7 1 T BW, Dose 12 hr PK sample Dose Dose and Begin 24 hr Urine collection 89 Collect 24 hr Urine and Dose 2 T BW, Dose 12 hr PK sample Dose Begin 24 hr Urine collection 96 Collect 24 hr Urine and Dose 3 T BW, Dose 12 hr PK sample Dose Begin 24 hr Urine collection Collect 24 hr Urine and Dose (last dose) 4 T BW No activity Begin 24 hr urine collection Collect 24 hr urine BW, Terminate, cardiac puncture blood draw, and collect kidneys, weigh and fix.

During both studies, pathological analysis is conducted as follows:

Specimen Anti- Specimen Clinical to be Method of Coag- to be Chemistry Method of Collected Collection ulant Analyzed Analyte Analysis Urine 24 hr NA Urine Creatinine/ AU480/ Metabolism Albumin ICL Elisa Cages Whole Tail Vein None Serum BUN AU480/ Blood Creatinine Sponsor Glucose/ Whole Cardiac None Serum BUN AU480/ Blood Puncture Creatinine Sponsor (Grps 1, 2, Glucose/ 3, 4 and 5; Cmpd Exp & and 6, 7, 8) Biomarker

Altogether, these results suggested that RNAs encoding BMP-7 variants effectively prevented the development of diabetic nephropathy in rats. In addition, these RNAs also effectively treated and restored kidney functions in rats already afflicted with diabetic nephropathy. RNA encoding BMP7 Variant A was particularly effective at preventing and treating diabetic nephropathy in rats.

Example 43: Transfection of Human Keratinocytes with RNA Encoding BDNF, BMP-2, BMP-6, IL-2, IL-6, IL-15, IL-22, LIF or FGF-21

100,000 human epidermal keratinocytes (HEK, Gibco) were plated in EpiLife+Supplement S7. Cells were transfected according to Example 3 with 2 μg of RNA encoding BDNF, BMP-2, BMP-6, IL-2, IL-6, IL-15, IL-22, LIF or FGF-21. 24 hours after transfection, the medium was sampled and secreted protein levels were measured using a human ELISA kit (see Table below) according the manufacturer's instructions. Secreted protein levels were determined by measuring 450 nm absorbance using a microplate reader (EMax Plus, Molecular Devices). Secreted protein levels are shown in FIG. 34, panels A-I.

Protein Part Number Vendor BDNF DBNT00 R&D Systems BMP-2 DBP200 R&D Systems BMP-6 ab99984 Abcam IL-2 D2050 R&D Systems IL-6 D6050 R&D Systems IL-15 D1500 R&D Systems IL-22 D2200 R&D Systems LIF DLF00 R&D Systems FGF-21 DF2100 R&D Systems

Example 44: Transfection of Human Keratinocytes with RNA Encoding IL-15 and/or IL-15RA

100,000 human epidermal keratinocytes (HEK, Gibco) were plated in EpiLife+Supplement S7. Cells were transfected according to Example 3 with 2 μg of RNA encoding IL-15 or 1 μg each of RNA encoding IL-15 and RNA encoding IL-15RA. 24 hours after transfection, the medium was sampled and secreted IL-15 levels were measured using a human IL-15 ELISA kit (D1500, R&D Systems) according the manufacturer's instructions. Secreted IL-15 levels were determined by measuring 450 nm absorbance using a microplate reader (EMax Plus, Molecular Devices). Secreted IL-15 levels are shown in FIG. 35. As demonstrated in FIG. 35, co-transfection of IL-15 and IL-15RA significantly increased secreted IL-15 levels compared to transfection with IL-15 alone.

Example 45: Pharmacokinetic Study Via Intradermal Injection in Rats

Studies were conducted to evaluate the responses of Sprague Dawley rats to intradermal administration of various RNAs. Specifically, 8-10 weeks old, female Sprague Dawley rats weighing about 200 g to about 350 g were used for this study. A total of 33 rats were tested, and the animals were assigned to study groups and treated as indicated in the Table below:

Dose Dose Volume Number of Test Dose Dose Level Concentration (μL/per Animals Group Group Color Article Route (μg) (μg/mL) injection)^(a) Females 1 White Control ID 4.0 20.0 200 3^(b) (NOVEPOEITIN) (4 × 50) 2 Yellow TA1 ID 4.0 20.0 200 3^(b) (IL2) (4 × 50) 3 Green TA2 ID 4.0 20.0 200 3^(b) (IL6) (4 × 50) 4 Blue TA3 ID 4.0 10.0 200 3^(b) (IL15) (4 × 50) 5 Red TA4 ID 4.0 20.0 200 3^(b) (IL15 + (10.0 each) (4 × 50) IL15RA) 6 Dark Grey TA5 ID 4.0 20.0 200 3^(b) (IL22) (4 × 50) 7 Purple TA6 ID 4.0 20.0 200 3^(b) (BMP2) (4 × 50) 8 Black TA7 ID 4.0 20.0 200 3^(b) (BDNF) (4 × 50) 9 White/Yellow TA8 ID 4.0 20.0 200 3^(b) (LIF) (4 × 50) 10 Green/Blue TA9 ID 4.0 20.0 200 3^(b) (PTH) (4 × 50) 11 Red/Dark Grey TA10 ID 4.0 20.0 200 3^(b) (FGF21) (4 × 50) ^(a)Total dose volume (μL/per animal) was constant. Each animal received four intradermal injections of 50 μL/per injection for a total of 200 μL per animal. ^(b)Animals, euthanized on Day 3 without examination or necropsy Intradermal = ID

For the study, the animals were treated with 4 ug of RNA. All groups were dosed via intradermal injection. Each dose was administered in four intradermal injections of 50 μL/injection for a total of 200 uL per animal. Injections occurred into previously marked sites near the midline of the dorsal lumbar area (upper left, upper right, lower left and lower right quadrants). Dose time (after the last injection) was recorded. Additional markings were made as needed to allow for identification of the dose site. Animals were administered with the RNAs on day 1 and euthanized on day 3. Clinical observations were made on the rats twice daily. Food consumption and body weight were also monitored.

During the study, approximately 1 ml of blood samples was collected from the jugular vein for pharmacokinetic analysis as follows:

Time Point PK^(a) Acclimation — Day 1 12 hours postdose Day 2 24 hours postdose Day 3 48 hours postdose ^(a)Time points for blood collection (n = 3 animals/Group/Time point) PK = Pharmacokinetics

Results indicate that following administration of RNAs encoding FGF21, IL15, IL15 and IL15R, IL6, IL22, and NOVEPOETIN, these proteins were readily detected in the blood with protein levels peaking at approximately 12 hours post injection (FIG. 39). Of note, the proteins tested in this study can be taken up by cells and tissues and/or can exert an effect near the site of expression without appreciable accumulation in systemic circulation.

Example 46: Wound-Healing Therapy Comprising RNA Encoding IL22

Primary human epidermal keratinocytes (HEKn-APF) were plated in 6-well plates coated with recombinant human Type-1 collagen at a density of 200,000 cells/well in keratinocytes medium (EpiLife+Supplement S7). After 24 h, cells were transfected with 2 μg/well RNA encoding IL22 synthesized according to Example 1 and 50 mM D-Glucose was added to the wells. The following day, a line was scratched in each well using a plastic 10 μL pipette tip. Wells were imaged at 0 h and 24 h after scratching. The size of the scratch was measured using at both times, and healing was determined by calculating the ration of the size of the scratch at 24 h to the size of the scratch at 0 h (FIG. 40).

Example 47: A1AT Gene Editing and Repair in Human Cells

RNA encoding gene-editing proteins targeting the following sequences in the A1AT gene was synthesized according to Example 1: L1: TAAGGCTGTGCTGACCATCG (SEQ ID NO: 611), R1: TAAAAACATGGCCCCAGCAG (SEQ ID NO: 612), and R2: TCTAAAAACATGGCCCCAGC (SEQ ID NO: 613). Cells were gene edited and gene-editing efficiency was measured according to Example 38 (FIG. 41). Additionally, cells were co-transfected with gene editing proteins targeting sequences L1 and R1 and a repair template comprising the sequence: cccctccaggccgtgcataaggctgtgctgaccatcgacgtcaaagggactgaagctgctggggccatgtttttagaggcc (SEQ ID NO: 614), and gene-repair efficiency was measured according to Example 38, using the AatII enzyme (FIG. 42). Another sample of cells was transfected with gene-editing proteins targeting the following sequences in the A1AT gene: L2: TGCCTGGTCCCTGTCTCCCT (SEQ ID NO: 615) and R3: TGTCTTCTGGGCAGCATCTC (SEQ ID NO: 616). Gene-editing efficiency was measured according to Example 38 (FIG. 43).

Example 48: Alpha-1-Antitrypsin Deficiency Therapy Comprising RNA Encoding Gene-Editing Proteins and a Repair Template

RNA encoding gene-editing proteins targeting the following sequences: L: TAAGGCTGTGCTGACCATCG (SEQ ID NO: 611) and R: TAAAAACATGGCCCCAGCAG (SEQ ID NO: 612) and a repair template comprising the sequence: RT: cccctccaggccgtgcataaggctgtgctgaccatcgacgagaaagggactgaagctgctggggccatgtttttagaggcc (SEQ ID NO: 617) are formulated with a vehicle according to the methods of the present invention and delivered to a subject afflicted with A1AT deficiency by intraportal administration, resulting in correction of the Z mutation in the subject's liver cells, reduction of polymerized Z protein accumulation in the subject's liver cells, increased secretion and serum levels of functional A1AT, and amelioration of one or more of the subject's symptoms.

Example 49: Friedrich's Ataxia Therapy Comprising RNA Encoding Gene-Editing Proteins

RNA encoding one or more gene-editing proteins capable of creating one or more double strand breaks in FXNA (SEQ ID NO: 582) and RNA encoding one or more gene-editing proteins capable of creating one or more double strand breaks in FXNB (SEQ ID NO: 583) are synthesized according to Example 1. RNA is formulated according to the methods of the present invention, and delivered to the heart of a patient with Friedrich's ataxia using a catheter. Target-sequence pairs are selected from: Pair 1: TCCCACACGTGTTATTTGGC (SEQ ID NO: 618) and TGGCAACCAATCCCAAAGTT (SEQ ID NO: 619); Pair 2: TAATAAATAAAAATAAAAAA (SEQ ID NO: 620) and TTGCCTATTTTTCCAGAGAT (SEQ ID NO: 621).

Example 50: Alpha-1-Antitrypsin Deficiency Therapy Comprising RNA Encoding Gene-Editing Proteins

RNA encoding one or more gene-editing proteins capable of creating one or more double strand breaks in A1AT_A (SEQ ID NO: 584) is synthesized according to Example 1. RNA is formulated according to the methods of the present invention, and is delivered to the liver of a patient with A1AT deficiency by intraportal injection.

Example 51: Alpha-1-Antitrypsin Deficiency Therapy Comprising RNA Encoding Gene-Editing Proteins and a Short Repair Template

RNA encoding one or more gene-editing proteins capable of creating one or more double strand breaks in A1AT_B (SEQ ID NO: 585) is synthesized according to Example 1. RNA and a single stranded DNA sequence containing the sequence: A1AT_RT (SEQ ID NO: 586) are formulated according to the methods of the present invention, and are delivered to the liver of a patient with A1AT deficiency by intraportal injection.

Example 52: Gene-Editing Proteins Comprising DNA-Binding and Deaminase Activity

RNA encoding a gene-editing protein comprising two or more repeat sequences, followed by: BASE_EDIT_FRONT (SEQ ID NO: 587), followed by any of: BASE_EDIT_ADA1 (SEQ ID NO: 588), BASE_EDIT_ADA2 (SEQ ID NO: 589), BASE_EDIT_ADA3 (SEQ ID NO: 590), BASE_EDIT_ADA4 (SEQ ID NO: 591), BASE_EDIT_CDA1 (SEQ ID NO: 592) and BASE_EDIT_CDA2 (SEQ ID NO: 593) is synthesized according to Example 1. The gene-editing protein is capable of correcting one or more mutations within 1 to 50 bases downstream of the target sequence.

Example 53: Gene Editing of A1AT, COL7A1, HBB, and PD-1

With reference to Example 1, Table 4, the following templates and targets were used:

SEQ ID Template Target Sequence NO: A1AT TALEN L TGCCTGGTCCCTGTCTCCCT 615 A1AT TALEN R TGTCTTCTGGGCAGCATCTC 616 A1AT RIBOSLICE L_A TGCCTGGTCCCTGTCTCCCT 615 A1AT RIBOSLICE L_B TGCCTGGTCCCTGTCTCCCT 615 A1AT RIBOSLICE R_A TGTCTTCTGGGCAGCATCTC 616 A1AT RIBOSLICE R_B TGTCTTCTGGGCAGCATCTC 616 COL7A1 exon 73 TALEN L TATTCCCGGGCTCCCAGGCA 622 COL7A1 exon 73 TALEN R TCTCCTGGCCTTCCTGCCTC 612 COL7A1 exon 73 rs3L 50A TATTCCCGGGCTCCCAGGCA 622 COL7A1 exon 73 rs3L 50B TATTCCCGGGCTCCCAGGCA 622 COL7A1 exon 73 rs3R 50A TCTCCTGGCCTTCCTGCCTC 612 COL7A1 exon 73 rs3R 50B TCTCCTGGCCTTCCTGCCTC 612 COL7A1 exon 73  TATTCCCGGGCTCCCAGGCA 622 TALEN L EA COL7A1 exon 73  TATTCCCGGGCTCCCAGGCA 622 TALEN L Het COL7A1 exon 73  TCTCCTGGCCTTCCTGCCTC 612 TALEN R EA COL7A1 exon 73  TCTCCTGGCCTTCCTGCCTC 612 TALEN R Het COL7A1 exon 73  TATTCCCGGGCTCCCAGGCA 622 TALEN L EA/Het COL7A1 exon 73  TCTCCTGGCCTTCCTGCCTC 612 TALEN R EA/Het HBB exon 1 TALEN L TGGTGCATCTGACTCCTGAG 623 HBB exon 1 TALEN R TCACCTTGCCCCACAGGGCA 624 PD-1 exon 1 TALEN L TCCAGGCATGCAGATCCCAC 625 PD-1 exon 1 TALEN R TTGTAGCACCGCCCAGACGA 626 TTR (forward (top)  TGCTGTCCGAGGCAGTCCTG  639; and reverse (bottom) TCAGCAGCCTTTCTGAACAC 640 TTR (forward (top)  TGTCCGAGGCAGTCCTGCCA  641; and reverse (bottom) TCAGCAGCCTTTCTGAACAC 642 TTR (forward (top)  TCCGAGGCAGTCCTGCCATC  643; and reverse (bottom) TCAGCAGCCTTTCTGAACAC 644 TTR (forward (top)  TGTCCGAGGCAGTCCTGCCA  645; and reverse (bottom) TCATCAGCAGCCTTTCTGAA 646 TTR (forward (top)  TCCGAGGCAGTCCTGCCATC  647; and reverse (bottom) TCATCAGCAGCCTTTCTGAA 648 TTR (forward (top)  TCCGAGGCAGTCCTGCCATC  649; and reverse (bottom) TGTCATCAGCAGCCTTTCTG 650

About 100,000 primary human neonatal epidermal keratinocytes were plated in EpiLife+Supplement S7 and transfected according to Example 3 with RNA encoding gene-editing proteins that target the sequences L: TGCCTGGTCCCTGTCTCCCT (SEQ ID NO: 615) and R: TGTCTTCTGGGCAGCATCTC (SEQ ID NO: 616), which were located approximately 75 bp from the Alpha-1-Antitrypsin (A1AT) start codon. Cells were gene edited, and gene-editing efficiency was measured as previously described. Results as shown in FIG. 46 demonstrate efficient gene-editing by the gene editing proteins.

About 100,000 primary human neonatal epidermal keratinocytes were plated in EpiLife+Supplement S7 and transfected according to Example 3 with RNA encoding gene-editing proteins that target the sequences L: TATTCCCGGGCTCCCAGGCA (SEQ ID NO: 622) and R: TCTCCTGGCCTTCCTGCCTC (SEQ ID NO: 612), which were located near the end of exon 73 of COL7A1. Cells were gene edited, and gene-editing efficiency was measured as previously described. Results as shown in FIG. 47 demonstrate efficient gene-editing by the gene editing proteins.

About 50,000 primary human neonatal epidermal keratinocytes were plated in EpiLife+Supplement S7 and transfected according to Example 3 with RNA encoding various gene-editing proteins that target the sequences L: TATTCCCGGGCTCCCAGGCA (SEQ ID NO: 622) and R: TCTCCTGGCCTTCCTGCCTC (SEQ ID NO: 612), which were located near the end of exon 73 of COL7A1. In particular, the gene-editing proteins comprised the endonuclease cleavage domain of FokI and variants thereof. The variants included a FokI variant with enhanced activity (S35P, K58E), a FokI heterodimer (i.e., L: Q103E, N113D, I116L; and R: E107K, H154R, 1155K), and a combination thereof (i.e., L: S35P, K58E, Q103E, N113D, I116L; and R: S35P, K58E, E107K, H154R, 1155K). Cells were gene edited, and gene-editing efficiency was measured as previously described. Results as shown in FIG. 48 demonstrate efficient gene-editing by the gene editing proteins.

About 100,000 primary human neonatal epidermal keratinocytes (animal-protein free) were plated in EpiLife+Supplement S7. Cells were transfected according to Example 3 with 2 μg RNA encoding the HBB exon 1 TALEN L and HBB exon 1 TALEN R gene-editing proteins (1 μg each). 48 hours after transfection, DNA was harvested and analyzed for gene editing (T7E1 assay; forward primer: gccaaggacaggtacggctgtcatc (SEQ ID NO: 627); reverse primer: cttgccatgagccttcaccttagggttg (SEQ ID NO: 628); product size: 518 nt; predicted band sizes: 300 nt, 218 nt). Results as shown in FIG. 61 demonstrate efficient gene-editing by the gene editing proteins.

About 100,000 primary human neonatal epidermal keratinocytes (animal-protein free) were plated in EpiLife+Supplement S7. Cells were transfected according to Example 3 with 2 μg RNA encoding the PD1 exon 1 TALEN L and PD1 exon 1 TALEN R gene-editing proteins (1 μg each). After 48 hours, DNA was harvested and analyzed for gene editing (T7E1 assay; forward primer: tcctctgtctccctgtctctgtctctctctc (SEQ ID NO: 594); reverse primer: ggacttgggccaggggaggag (SEQ ID NO: 595); product size: 612 nt; predicted band sizes: 349 nt, 263 nt).

Results as shown in FIG. 62 demonstrate efficient gene-editing by the gene editing proteins.

Example 54: Transfection of Primary Human Keratinocytes with RNA Encoding GDF15, IFKB, KRT5, KRT14, SOD3, or hESM1

As shown in FIG. 49, 100,000 primary human neonatal epidermal keratinocytes were plated in EpiLife+Supplement S7. Cells were transfected according to Example 3 with 2 μg of RNA encoding hGDF15. Following transfection, the culture media was sampled at various time points and analyzed for hGDF15 levels using ELISA (R&D DGD150) according to manufacturer's instructions. FIG. 49 demonstrates that hGDF15 levels were upregulated in a time-dependent manner following transfection.

As shown in FIG. 50, 100,000 primary human neonatal epidermal keratinocytes were plated in EpiLife+Supplement S7. Cells were transfected according to Example 3 with 2 μg of RNA encoding IFKB or an IFKB variant (S32A and S36A). Following transfection, the culture media was sampled at various time points and analyzed for IFKB levels using ELISA (Abcam, ab176644) according to manufacturer's instructions. FIG. 50 demonstrates increased levels IFKB or the IFKB variant following transfection.

As shown in FIG. 51, 100,000 primary human neonatal epidermal keratinocytes were plated in 6-well plates in EpiLife+Supplement S7. Cells were transfected according to Example 3 with 2 μg of RNA encoding KRT5 or KRT14 (or GFP tagged versions of KRT5 or KRT14). Following transfection, GFP imaging was carried out to determine KRT5 or KRT14 expression. FIG. 51 demonstrates efficient transfection and increased levels KRT5-GFP or KRT14-GFP following transfection.

As shown in FIG. 52, 20,000 primary human neonatal epidermal keratinocytes were plated in 24-well plates in EpiLife+Supplement S7. Cells were transfected according to Example 3 with 0.2 μg of RNA encoding SOD3. Following transfection, cells were fixed and stained for 24 hours with a 1:100 dilution of NBP2-38493 (Novus) rabbit anti human SOD3 primary antibody and a 1:1000 dilution of 488 donkey anti-rabbit secondary antibody. Results demonstrate efficient transfection and robust levels of SOD3 in transfected cells.

As shown in FIG. 60, 200,000 primary human neonatal epidermal keratinocytes (animal-protein free) were plated in 6-well plates in EpiLife+Supplement S7. Cells were transfected according to Example 3 with 2 μg RNA encoding hESM1. 52 hours after transfection, the culture medium was analyzed for ESM1 by ELISA (Abcam ab213776). Results demonstrate efficient transfection and robust levels of hESM1 in transfected cells.

Example 55: In Vivo Targeting of GDF15 (MIC1)

Studies were conducted to evaluate the responses of Zucker-obese (ZDF) rats to administration of RNA encoding hGDF15. The Zucker-obese rats (ZDF) are a well-studied obesity model that mimics many aspects of the human disease. Specifically, 8-10 weeks old male rats were used for this study. A total of 15 ZDF and 15 wildtype control Sprague-Dawley rats were tested, and the animals were assigned to study groups and treated as indicated in the Table below:

Dose Group Rat Test Level Concentration Volume^(a) Number Group Color Species Article Route (μg) (μg/mL) (μL) of Males 1 White Sprague- Control Intradermal 0 0 50 per site; 5 Dawley 500 total 2 Yellow Sprague- FG-02 10X 10 50 per site; 10 Dawley (i.e., RNA (0.5 μg/ 500 total encoding injection; 5 GDF15) μg total) 3 Green ZDF Control 0 0 50 per site; 5 500 total 4 Blue ZDF FG-02 10X 10 50 per site; 10 (i.e., RNA (0.5 μg/ 500 total encoding injection; 5 GDF15) μg total) ^(a)Note: Total dose volume (μL/per animal) was constant

Groups 1 to 4 were dosed by intradermal (ID) injection. Each dose was administered on the back in ten ID injections of 50 μL/injection for a total of 500 uL per animal.

Each dose site was as follows:

RNA or control was administered once weekly (Days 1, 8, and 15), with the first dose being on Day 1, and the last dose on Day 15. Animals were euthanized, without examination on Day 44.

Blood was collected from the rats according to the schedule below and used for serum chemistry and pharmacokinetics analysis:

Time Point (Study Week) Serum Chemistry PK Acclimation — — (Week −2) Dosing 2x Day 1 (1x), 2 (1x), 3 (1x), 4 (1x), (Week 1) and 5 (1x) Dosing 2x Day 9^(c) (Week 2) Dosing 2x Day 16^(c) (Week 3) Dosing 2x Day 23 (Week 4) Dosing 2x — (Week 5) Dosing 2x — (Week 6) Dosing 1x — (Week 7) — = Not applicable PK = Pharmacokinetics x = Number of times procedure performed ^(c)Sample collected 24 hours postdose

Serum chemistry analysis was carried out using an AU680 analyzer for the following: Albumin, Alkaline Phosphatase, Alanine Aminotransferase, Aspartate Aminotransferase, Total Bilirubin, Calcium, Total Cholesterol, Creatine Kinase, Creatinine, Glucose, Inorganic Phosphorus, Total Protein, Triglyceride, Sodium, Potassium, Chloride, Globulin, Albumin/Globulin Ratio, and Blood Urea Nitrogen. Results for changes in the serum levels of ALT, AST, total cholesterol, triglycerides, and glucose are provided in FIGS. 53-58. As shown in the FIG. 58, administration of RNA encoding GDF15 into ZDF rats resulted in reduced ALT, AST, and Glucose levels but elevated total cholesterol and triglyceride levels in the serum. These results suggest that diabetic obese animals treated with RNA encoding GDF15 exhibited improved liver health, as indicated by decreased serum levels of ALT and AST, improved (lower) blood glucose levels, and lipid mobilization, as indicated by increased serum cholesterol and triglyceride levels. These results therefore suggest that RNA encoding GDF15 may be used to treat, inter alia, diabetes, obesity, fatty liver, and other diseases that include liver inflammation, elevated blood glucose, and/or abnormal lipid metabolism.

The serum levels of GDF15 in the treated rats were confirmed as shown in FIG. 59. As shown, robust GDF15 levels were observed even at day 16 (i.e., day 15+24 h) in both ZDF and control Sprague-dawley rats injected with RNA encoding GDF15.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporated by reference in their entireties. 

1-179. (canceled)
 180. A composition comprising a cell derived from a subject having an alpha-1 antitrypsin (A1AT) deficiency, comprising: (a) a synthetic RNA encoding a gene-editing protein capable of creating a single-strand or double-strand break in an A1AT gene, the gene-editing protein comprising: (i) a DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 629) and is between 36 and 39 amino acids long, wherein: “v” is Q, D or E, “w” is S or N, “x” is H, N, or I, “y” is D, A, I, N, G, H, K, S, or null, and “z” is GGKQALETVQRLLPVLCQD (SEQ ID NO: 630) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 631); and (ii) a nuclease domain comprising a catalytic domain of a nuclease, and (b) a single-strand or double-strand break in an A1AT gene, the single-strand or double-strand break being caused by the gene-editing protein.
 181. The composition of claim 180, wherein the synthetic RNA comprises one or more non-canonical nucleotides that avoid substantial cellular toxicity.
 182. The composition of claim 180, wherein the composition is suitable for administration to the liver.
 183. The composition of claim 180, wherein the composition is suitable for intraportal injection.
 184. The composition of claim 180, wherein the nuclease domain is capable of forming a dimer with another nuclease domain.
 185. The composition of claim 180, wherein the gene-editing protein, either alone or in combination with one or more other molecules, confers correction of the Z mutation.
 186. The composition of claim 180, wherein the gene-editing protein, either alone or in combination with one or more other molecules, confers reduction of polymerized Z protein accumulation.
 187. The composition of claim 180, wherein the gene-editing protein, either alone or in combination with one or more other molecules, confers an increase in secretion and/or serum levels of functional A1AT.
 188. The composition of claim 180, wherein the gene-editing protein targets a sequence selected from: TAAGGCTGTGCTGACCATCG (SEQ ID NO: 611), TAAAAACATGGCCCCAGCAG (SEQ ID NO: 612), TCTAAAAACATGGCCCCAGC (SEQ ID NO: 613), TGCCTGGTCCCTGTCTCCCT (SEQ ID NO: 615) and TGTCTTCTGGGCAGCATCTC (SEQ ID NO: 616).
 189. A composition comprising a cell derived from a subject having an alpha-1 antitrypsin (A1AT) deficiency, comprising: (a) a synthetic RNA encoding a gene-editing protein capable of creating a single-strand or double-strand break in A1AT_A (SEQ ID NO: 584) and/or a synthetic RNA encoding a gene-editing protein capable of creating a single-strand or double-strand break in A1AT_B (SEQ ID NO: 585), wherein the gene-editing protein comprises: (i) a DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 629) and is between 36 and 39 amino acids long, wherein: “v” is Q, D or E, “w” is S or N, “x” is H, N, or I, “y” is D, A, I, N, G, H, K, S, or null, and “z” is GGKQALETVQRLLPVLCQD (SEQ ID NO: 630) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 631); and (ii) a nuclease domain comprising a catalytic domain of a nuclease, and (b) a single-strand or double-strand break in an A1AT gene, the single-strand or double-strand break being caused by the gene-editing protein.
 190. The composition of claim 189, comprising: (a) the synthetic RNA encoding the gene-editing protein capable of creating a single-strand or double-strand break in A1AT_A (SEQ ID NO: 584), and (b) the synthetic RNA encoding the gene-editing protein capable of creating a single-strand or double-strand break in A1AT_B (SEQ ID NO: 585).
 191. The composition of claim 189, wherein the synthetic RNA comprises one or more non-canonical nucleotides that avoid substantial cellular toxicity.
 192. The composition of claim 189, wherein the composition is suitable for administration to the liver.
 193. The composition of claim 189, wherein the composition is suitable for intraportal injection.
 194. The composition of claim 189, wherein the nuclease domain is capable of forming a dimer with another nuclease domain.
 195. The composition of claim 189, wherein the gene-editing protein, either alone or in combination with one or more other molecules, confers correction of the Z mutation.
 196. The composition of claim 189, wherein the gene-editing protein, either alone or in combination with one or more other molecules, confers reduction of polymerized Z protein accumulation.
 197. The composition of claim 189, wherein the gene-editing protein, either alone or in combination with one or more other molecules, confers an increase in secretion and/or serum levels of functional A1AT.
 198. The composition of claim 189, wherein the gene-editing protein targets a sequence selected from: TAAGGCTGTGCTGACCATCG (SEQ ID NO: 611), TAAAAACATGGCCCCAGCAG (SEQ ID NO: 612), TCTAAAAACATGGCCCCAGC (SEQ ID NO: 613), TGCCTGGTCCCTGTCTCCCT (SEQ ID NO: 615) and TGTCTTCTGGGCAGCATCTC (SEQ ID NO: 616). 